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In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

Komuta Y, Ishii T, Kaneda M, Ueda Y, Miyamoto K, Toyoda M, Umezawa A, Seko Y - Biol Open (2016)

Bottom Line: We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions.In functional studies, a light-induced inward current was detected in some CRX-transduced cells.Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs.

View Article: PubMed Central - PubMed

Affiliation: Visual Functions Section, Department of Rehabilitation for Sensory Functions, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Saitama 359-8555, Japan.

No MeSH data available.


Related in: MedlinePlus

PBMCs transduced with CRX via Sendai virus (SeV) expressed photoreceptor-related genes. (A) Comparison between induced photoreceptor-related genes in CRX-transduced PBMCs and those in commercially available human dermal fibroblasts prepared by using SeV or retrovirus vectors. All PBMCs collected from the three volunteers were analyzed, and showed figures of blue opsin was detected from No. 2 PBMCs sample, red/green opsin was from No. 3 sample, rhodopsin was from No. 1 sample (nested PCR). Bands at the far right in the rhodopsin lanes show positive controls from the Y79 sample (positive control). CRX (total): total expression of CRX (35 PCR cycles); CRX (endo): endogenous expression of CRX (40 PCR cycles); G3PDH: housekeeping gene as internal control. (B) Immunocytochemistry and populations of cells expressing blue opsin (green) or HN (red). White arrows indicate a representative double-stained cell (enlarged in the left square). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. Populations of blue opsin- or HN-positive cells were displayed in graphs (bottom). (n=4, **P<0.01; Student's t-test). (C) Temporal patterns of detected photoreceptor-related genes PDE6H, blue opsin, and SAG determined by real-time PCR. No. 2 PBMCs were analyzed for 6 h, 1 day, 2 days, 3 days, 5 days, 1 week and 2 weeks after CRX transduction. Samples from controls at 6 h were used as references (n=3; *P<0.05, **P<0.01; Dunnett's test). (D) Induced endogenous CRX expression. Specific primer sets were designed to distinguish endogenous from exogenous expression of CRX. Endogenous CRX expression was detected in CRX-transduced PBMCs at 1 week after transduction (endo: endogenous CRX, G3: G3PDH). All error bars represent s.d.
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BIO016477F1: PBMCs transduced with CRX via Sendai virus (SeV) expressed photoreceptor-related genes. (A) Comparison between induced photoreceptor-related genes in CRX-transduced PBMCs and those in commercially available human dermal fibroblasts prepared by using SeV or retrovirus vectors. All PBMCs collected from the three volunteers were analyzed, and showed figures of blue opsin was detected from No. 2 PBMCs sample, red/green opsin was from No. 3 sample, rhodopsin was from No. 1 sample (nested PCR). Bands at the far right in the rhodopsin lanes show positive controls from the Y79 sample (positive control). CRX (total): total expression of CRX (35 PCR cycles); CRX (endo): endogenous expression of CRX (40 PCR cycles); G3PDH: housekeeping gene as internal control. (B) Immunocytochemistry and populations of cells expressing blue opsin (green) or HN (red). White arrows indicate a representative double-stained cell (enlarged in the left square). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. Populations of blue opsin- or HN-positive cells were displayed in graphs (bottom). (n=4, **P<0.01; Student's t-test). (C) Temporal patterns of detected photoreceptor-related genes PDE6H, blue opsin, and SAG determined by real-time PCR. No. 2 PBMCs were analyzed for 6 h, 1 day, 2 days, 3 days, 5 days, 1 week and 2 weeks after CRX transduction. Samples from controls at 6 h were used as references (n=3; *P<0.05, **P<0.01; Dunnett's test). (D) Induced endogenous CRX expression. Specific primer sets were designed to distinguish endogenous from exogenous expression of CRX. Endogenous CRX expression was detected in CRX-transduced PBMCs at 1 week after transduction (endo: endogenous CRX, G3: G3PDH). All error bars represent s.d.

Mentions: Expression of photoreceptor-related genes was examined by RT-PCR 7 days after transduction of the CRX gene alone by retrovirus vectors or Sendai virus vectors (SeV vectors) in PBMCs isolated from the blood of three healthy donors, designated No. 1-3. We found that the CRX gene was effective in inducing photoreceptor-related genes, blue opsin and red/green opsin, in the PBMCs. After transduction of the CRX gene by SeV-CRX at 20 or 50 MOI, PBMCs efficiently expressed the blue opsin and red/green opsin genes (Fig. 1A). When PBMCs were transduced with retrovirus vectors, the blue opsin gene was not detected. The red/green opsin gene was specifically expressed in cone-photoreceptor cells. Compared with that observed in PBMCs, human dermal fibroblasts expressed these photoreceptor-related genes at a much lower level following transduction of CRX alone via retrovirus or SeV vectors. However, rhodopsin was not detected following transduction of CRX alone.Fig. 1.


In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

Komuta Y, Ishii T, Kaneda M, Ueda Y, Miyamoto K, Toyoda M, Umezawa A, Seko Y - Biol Open (2016)

PBMCs transduced with CRX via Sendai virus (SeV) expressed photoreceptor-related genes. (A) Comparison between induced photoreceptor-related genes in CRX-transduced PBMCs and those in commercially available human dermal fibroblasts prepared by using SeV or retrovirus vectors. All PBMCs collected from the three volunteers were analyzed, and showed figures of blue opsin was detected from No. 2 PBMCs sample, red/green opsin was from No. 3 sample, rhodopsin was from No. 1 sample (nested PCR). Bands at the far right in the rhodopsin lanes show positive controls from the Y79 sample (positive control). CRX (total): total expression of CRX (35 PCR cycles); CRX (endo): endogenous expression of CRX (40 PCR cycles); G3PDH: housekeeping gene as internal control. (B) Immunocytochemistry and populations of cells expressing blue opsin (green) or HN (red). White arrows indicate a representative double-stained cell (enlarged in the left square). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. Populations of blue opsin- or HN-positive cells were displayed in graphs (bottom). (n=4, **P<0.01; Student's t-test). (C) Temporal patterns of detected photoreceptor-related genes PDE6H, blue opsin, and SAG determined by real-time PCR. No. 2 PBMCs were analyzed for 6 h, 1 day, 2 days, 3 days, 5 days, 1 week and 2 weeks after CRX transduction. Samples from controls at 6 h were used as references (n=3; *P<0.05, **P<0.01; Dunnett's test). (D) Induced endogenous CRX expression. Specific primer sets were designed to distinguish endogenous from exogenous expression of CRX. Endogenous CRX expression was detected in CRX-transduced PBMCs at 1 week after transduction (endo: endogenous CRX, G3: G3PDH). All error bars represent s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920181&req=5

BIO016477F1: PBMCs transduced with CRX via Sendai virus (SeV) expressed photoreceptor-related genes. (A) Comparison between induced photoreceptor-related genes in CRX-transduced PBMCs and those in commercially available human dermal fibroblasts prepared by using SeV or retrovirus vectors. All PBMCs collected from the three volunteers were analyzed, and showed figures of blue opsin was detected from No. 2 PBMCs sample, red/green opsin was from No. 3 sample, rhodopsin was from No. 1 sample (nested PCR). Bands at the far right in the rhodopsin lanes show positive controls from the Y79 sample (positive control). CRX (total): total expression of CRX (35 PCR cycles); CRX (endo): endogenous expression of CRX (40 PCR cycles); G3PDH: housekeeping gene as internal control. (B) Immunocytochemistry and populations of cells expressing blue opsin (green) or HN (red). White arrows indicate a representative double-stained cell (enlarged in the left square). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. Populations of blue opsin- or HN-positive cells were displayed in graphs (bottom). (n=4, **P<0.01; Student's t-test). (C) Temporal patterns of detected photoreceptor-related genes PDE6H, blue opsin, and SAG determined by real-time PCR. No. 2 PBMCs were analyzed for 6 h, 1 day, 2 days, 3 days, 5 days, 1 week and 2 weeks after CRX transduction. Samples from controls at 6 h were used as references (n=3; *P<0.05, **P<0.01; Dunnett's test). (D) Induced endogenous CRX expression. Specific primer sets were designed to distinguish endogenous from exogenous expression of CRX. Endogenous CRX expression was detected in CRX-transduced PBMCs at 1 week after transduction (endo: endogenous CRX, G3: G3PDH). All error bars represent s.d.
Mentions: Expression of photoreceptor-related genes was examined by RT-PCR 7 days after transduction of the CRX gene alone by retrovirus vectors or Sendai virus vectors (SeV vectors) in PBMCs isolated from the blood of three healthy donors, designated No. 1-3. We found that the CRX gene was effective in inducing photoreceptor-related genes, blue opsin and red/green opsin, in the PBMCs. After transduction of the CRX gene by SeV-CRX at 20 or 50 MOI, PBMCs efficiently expressed the blue opsin and red/green opsin genes (Fig. 1A). When PBMCs were transduced with retrovirus vectors, the blue opsin gene was not detected. The red/green opsin gene was specifically expressed in cone-photoreceptor cells. Compared with that observed in PBMCs, human dermal fibroblasts expressed these photoreceptor-related genes at a much lower level following transduction of CRX alone via retrovirus or SeV vectors. However, rhodopsin was not detected following transduction of CRX alone.Fig. 1.

Bottom Line: We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions.In functional studies, a light-induced inward current was detected in some CRX-transduced cells.Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs.

View Article: PubMed Central - PubMed

Affiliation: Visual Functions Section, Department of Rehabilitation for Sensory Functions, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Saitama 359-8555, Japan.

No MeSH data available.


Related in: MedlinePlus