In vitro differentiation capacity of telomerase immortalized human RPE cells

Lakshmi Rambhatla, Choy Pik Chiu, Randolph D Glickman, Cheryl Rowe-Rendleman

Research output: Contribution to journalArticle

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Abstract

PURPOSE. To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells. METHODS. Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation. RESULTS. Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5′-bromo-2′-deoxyuridine (BrdU) up-take and positive reactivity with antibodies to cellular retinal-dehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G1 after 8 weeks of serum deprivation. CONCLUSIONS. Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

Original languageEnglish (US)
Pages (from-to)1622-1630
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number5
StatePublished - 2002
Externally publishedYes

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Retinal Pigments
Telomerase
Epithelial Cells
Serum
In Vitro Techniques
Vimentin
Bromodeoxyuridine
Growth
Keratins
Population
Cell Differentiation
Cell Cycle
Carrier Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

In vitro differentiation capacity of telomerase immortalized human RPE cells. / Rambhatla, Lakshmi; Chiu, Choy Pik; Glickman, Randolph D; Rowe-Rendleman, Cheryl.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 5, 2002, p. 1622-1630.

Research output: Contribution to journalArticle

Rambhatla, Lakshmi ; Chiu, Choy Pik ; Glickman, Randolph D ; Rowe-Rendleman, Cheryl. / In vitro differentiation capacity of telomerase immortalized human RPE cells. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 5. pp. 1622-1630.
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PY - 2002

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N2 - PURPOSE. To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells. METHODS. Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation. RESULTS. Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5′-bromo-2′-deoxyuridine (BrdU) up-take and positive reactivity with antibodies to cellular retinal-dehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G1 after 8 weeks of serum deprivation. CONCLUSIONS. Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

AB - PURPOSE. To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells. METHODS. Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation. RESULTS. Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5′-bromo-2′-deoxyuridine (BrdU) up-take and positive reactivity with antibodies to cellular retinal-dehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G1 after 8 weeks of serum deprivation. CONCLUSIONS. Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

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