Influence of bicarbonate and insulin on pigment synthesis by cultured adult human retinal pigment epithelial cells

It is usually assumed that the adult retinal pigment epithelium.(RPE) of humans and other mammals has negligible capacity to synthesize melanin granules.Consistent with this idea is the observation that human RPE cells that have divided extensively in culture lose endogenous pigment by dilution and do not synthesize new pigment in vitro (Flood, Gouras and Kjeldbye, 1980).However, adult lapine (Laties and Lerner, 1975) and bovine (Dryja et al., 1978) RPE contains tyrosinase activity, and RPE cultured from 4-6-month-old pigs synthesizes pigment granules in vitro (Dorey, Torres and Swart, 1990).Recently, during studies in this laboratory unrelated to pigment synthesis, cultures of aged adult human RPE maintained with Dulbecco's Modified Eagle's (DME) medium supplemented with insulin were observed to contain foci of newly divided cells that became intensely pigmented.Pigment synthesis by adult human RPE cultured in a DME-based medium has also been observed by others (C.K. Dorey, pers.commun.).Since de novo pigment synthesis by RPE cultured from aged human donors had not previously been observed with a number of other media [minimum essential medium (MEM), medium 199, Ham's F-10 nutrient solution, or PM medium ; Flood et al., 1980 ; Edwards and Kurtz, unpubl.ohs.], the result with DME supplemented with insulin prompted experiments to define more precisely the medium requirements for pigment synthesis.A possibly related observation is the report that increased bicarbonate levels led to earlier pigment synthesis by B16 melanoma cells (Laskin et al., 1980).In this report it is, shown that cultured human RPE from aged donors synthesize pigment when maintained in DME but not in other media, that an elevated bicarbonate level is a required feature of DME for this synthesis under the conditions of these experiments, and that insulin added to DME significantly shortens the in vitro time required for this synthesis.Human autopsy eye of donors 64 or more years of age were obtained through the National Disease Research Interchange, Philadelphia.RPE cells were isolated as previously described (Edwards, 1982) suspended in PM medium (Pfeffer et al., 1986 ;Edwards, Adler and Claycomb, 1991) that included 10 pug ml-' bovine insulin (Collaborative Research, Bedford, MA) and 20 pg ml-' gentamicin, and seeded at 5 or 10 x lo3 cells cm+ in 4-cm2 12-cluster wells (Costar, Cambridge, MA) or in 25-cm2 flasks (Falcon, Oxnard, CA).Additional cells were seeded at the same densities in 96-cluster wells for immunocytochemical 00144835/91/l 10681+04 $03.