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Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

Poon MW, He J, Fang X, Zhang Z, Wang W, Wang J, Qiu F, Tse HF, Li W, Liu Z, Lian Q - PLoS ONE (2015)

Bottom Line: Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%).Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs.This cell type may also have advantages in retinal pigmented epithelial differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR, China; The HKU Shenzhen Institute of Research and Innovation, The University of Hong Kong, Hong Kong SAR, China; Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

ABSTRACT
A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

No MeSH data available.


Related in: MedlinePlus

Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues.(A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4, TERT, NANOG, GDF3, REX1, ESG1, OCT4A, SOX2, KLF4 and C-MYC and Beta- actin. Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3&4: OEC1&2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.
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pone.0131288.g001: Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues.(A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4, TERT, NANOG, GDF3, REX1, ESG1, OCT4A, SOX2, KLF4 and C-MYC and Beta- actin. Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3&4: OEC1&2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.

Mentions: We recently isolated conjunctival epithelial cells (OECs) and conjunctival stromal cells (OSCs) from individual conjunctival specimen in conditioned cultural medium (S5 Fig). Using a characterized antibody (Santa Cruz Biotechnology, CA. Cat No: SC-8626) that specifically targeted the pluripotency factor OCT4A [17], we detected OCT4A expression mainly in the basal layer of conjunctival epithelium, but not in the stromal cell layers (Fig 1A). Immunofluorescence staining also disclosed that OCT4A is expressed mostly in conjunctival derived OECs, not in OSCs (Fig 1B). Similarly, pluripotency reprogramming factors, OCT4A and SOX2 are highly expressed in the basal layer of conjunctival epithelium rather than the stromal cell layers (Fig 1Ci). This is consistent with previous reports that OCT4 and SOX2 are expressed in corneal and conjunctival epithelium [18–20] In addition, isoform OCT4B was found widely expressed in OECs, OECiPSCs, OSCs, OSCiPSCs, skin fibroblast IMR90 (ATCC, CCL-186) and IMR90iPSCs (Fig 1Cii). Retrovirus-mediated reprograming factor OCT4 can be detected in OECiPSCs and OSCiPSCs, but not in OECs, OSCs and IMR90 somatic cells. (Fig 1Cii; S3 Fig for primers). Western blotting revealed expression of OCT4A in both human and mouse OECs at approximately 10%-15% of the protein level found in iPSCs (Fig 1D and 1Ei). Meanwhile, another reprogramming factor, SOX2, was also detected in mouse and human OECs (Fig 1D and 1Eii).


Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

Poon MW, He J, Fang X, Zhang Z, Wang W, Wang J, Qiu F, Tse HF, Li W, Liu Z, Lian Q - PLoS ONE (2015)

Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues.(A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4, TERT, NANOG, GDF3, REX1, ESG1, OCT4A, SOX2, KLF4 and C-MYC and Beta- actin. Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3&4: OEC1&2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.
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pone.0131288.g001: Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues.(A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4, TERT, NANOG, GDF3, REX1, ESG1, OCT4A, SOX2, KLF4 and C-MYC and Beta- actin. Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3&4: OEC1&2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.
Mentions: We recently isolated conjunctival epithelial cells (OECs) and conjunctival stromal cells (OSCs) from individual conjunctival specimen in conditioned cultural medium (S5 Fig). Using a characterized antibody (Santa Cruz Biotechnology, CA. Cat No: SC-8626) that specifically targeted the pluripotency factor OCT4A [17], we detected OCT4A expression mainly in the basal layer of conjunctival epithelium, but not in the stromal cell layers (Fig 1A). Immunofluorescence staining also disclosed that OCT4A is expressed mostly in conjunctival derived OECs, not in OSCs (Fig 1B). Similarly, pluripotency reprogramming factors, OCT4A and SOX2 are highly expressed in the basal layer of conjunctival epithelium rather than the stromal cell layers (Fig 1Ci). This is consistent with previous reports that OCT4 and SOX2 are expressed in corneal and conjunctival epithelium [18–20] In addition, isoform OCT4B was found widely expressed in OECs, OECiPSCs, OSCs, OSCiPSCs, skin fibroblast IMR90 (ATCC, CCL-186) and IMR90iPSCs (Fig 1Cii). Retrovirus-mediated reprograming factor OCT4 can be detected in OECiPSCs and OSCiPSCs, but not in OECs, OSCs and IMR90 somatic cells. (Fig 1Cii; S3 Fig for primers). Western blotting revealed expression of OCT4A in both human and mouse OECs at approximately 10%-15% of the protein level found in iPSCs (Fig 1D and 1Ei). Meanwhile, another reprogramming factor, SOX2, was also detected in mouse and human OECs (Fig 1D and 1Eii).

Bottom Line: Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%).Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs.This cell type may also have advantages in retinal pigmented epithelial differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR, China; The HKU Shenzhen Institute of Research and Innovation, The University of Hong Kong, Hong Kong SAR, China; Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

ABSTRACT
A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

No MeSH data available.


Related in: MedlinePlus