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Enhancing RPE-derivation from Pluripotent Stem Cells

Original article from Stem Cells Translational Medicine

"A Simple and Scalable Process for the Differentiation of Retinal Pigment Epithelium From Human Pluripotent Stem Cells" and "Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium"

The high prevalence of blindness caused by age-related macular degeneration (AMD) (Gehrs et al), due to damaged or dysfunctional retinal pigment epithelium (RPE) cells (Khandhadia et al) has led to the use of pluripotent stem cells to derive RPE for transplantation.   Various studies have shown that RPE can be derived from human embryonic stem cells (hESCs) (Klimanskaya et al) and human induced pluripotent stem cells (hiPSCs) (Buchholz et al, Hirami et al, Meyer et al and Osakada et al) and a human clinical trial of hESC-RPE cell transplantation is currently under way (Schwartz et al).   However, techniques used so far are problematic for large scale production of consistent high quality cells.   Now in two studies published in Stem Cells Translational Medicine, advancements in differentiation protocols are presented.   In the first study, researchers from the group of Donald J. Zack at the Johns Hopkins University School of Medicine Baltimore, Maryland, USA have described a less labour-intensive myosin inhibitor-mediated differentiation protocol which, after enrichment, leads to a highly pure population of cells which display many characteristics of native RPE cells (Maruotti et al).   In the latter study researchers from the laboratories of Peter J. Coffey and Dennis O. Clegg at the Neuroscience Research Institute and the Center for the Study of Macular Degeneration at the University of California, Santa Barbara, USA describe their work into the modification of current protocols by the addition of retinal induction factors and other factors at specific times giving an increased efficiency of RPE derivation at earlier time points (Buchholz et al).

Culture of human pluripotent stem cells (hPSCs: hiPSCs and hESCs) in the study by Maruotti et al was undertaken based on a previous report (Walker et al) using the clonal propagation of cells in the presence of the myosin inhibitor blebbistatin on Matrigel or vitronectin peptide-acrylate surface (VN-PAS), which allows the maintenance of a high level of pluripotent marker expression and a stable karyotype.  Differentiation was induced in monolayer cultures at high density for 8 days followed by the addition of RPE differentiation medium (described in detail in the original article).   After several weeks, RPE-like cells were generated with pigmented colonies appearing at 25-30 days of differentiation.   During this time, pluripotency-associated factors decreased while RPE-associated factors (PAX6, MITF1, BEST1, RPE56, RLBP1, but not OTX2) increased, and by 50 days RPE65+ cells constituted around 16% of differentiating hiPSC and hESC cultures.   Culture of these RPE65+ cells, followed by further selection and culture in RPE medium, allowed for the cultivation of a monolayer of lightly pigmented cells with cobblestone morphology, with few other contaminating cell types.   Maturation for another 20 days gave a strongly pigmented monolayer culture of cells with polygonal morphology which also exhibited dome structures indicative of active fluid transport known to occur in mature tight junction-bearing RPE cells (Strauss).    Additionally, these cells expressed high levels of MITF, OTX2, RLBP1 and RPE65, membrane situated BEST1 and the tight junction protein ZO-1 (detected by immunohistochemistry) and high mRNA levels of MITF, OTX2, BEST1, EMMPRIN, melanogenesis genes (TYR and PMEL), retinoid recycling genes (RLBP1, CRALBP and LRAT), PEDF and MERTK, all of which are known to be associated with RPE.   No difference was observed in mRNA expression between cells grown on VN-PAS compared with Matrigel, or between cells grown by serial passaging or manual picking. Overall, hPSC-RPE cells displayed the appropriate morphology and gene expression patterns - although some gene expression differences were noted upon comparison of hPSC-RPE with foetal RPE or the M1 primary line of adult RPE cells, consistent with previous reports (Liao et al).   The functionality of RPE cells partly entails phagocytosis of outer segments shed by photoreceptors and the secretion of growth factors, such as VEGF, in a polarized manner (Strauss).   hPSC-RPE layers showed evidence of both of these functions and a phagocytosis assay showed similar levels of functionality between serially passaged and manually picked RPE cells.   Finally, survival in vivo was assessed by injection of fluorescently labelled hESC-RPE cells into the subretinal space of albino NOD-scid mice.   Analysis at one week demonstrated numerous pigmented clusters and that hESC-RPE cells had formed round masses in the subretinal space.  Importantly, evidence for phagocytosis of mouse photoreceptor outer segments by hESC-RPE cells was demonstrated.

Buchholz et al based their differentiation protocols on previous reports finding that nicotinamide could increase differentiation of RPE from hPSCs (Idelson et al).   Cell clumps dissected from hESC colonies in the presence of nicotinamide, IGF1, NOGGIN, DKK1, and bFGF on Matrigel led to a significantly larger drop in pluripotency associated gene expression than without nicotinamide.   Furthermore, early neural/eye field markers (LHX2 and RAX) were further increased, while addition of nicotinamide also altered the morphology of hESCs towards what the authors call a radial/rosette morphology more rapidly.   To direct the cell towards RPE, all previously added factors were removed and replaced with Activin A, SU5402 (FGFR1 inhibitor), and vasoactive intestinal peptide (VIP) from days 4 to 6.   Activin A alone and in combination with SU5402 to day 10 reduced the expression of RAX while VIP, known to speed up maturation of cultured primary RPE by increasing intracellular cAMP and activating pp60(c-src) (Koh), increased the expression of RPE marker genes (MITF, TYR, and PEDF).   Concomitantly, pigmentation increased alongside the appearance of sheets of cells with cobblestone morphology and distinct borders.   Further culture with Activin A, SU5402, and VIP to day 14 led to the borders becoming more defined with cells beginning to pigment within.   In these cells, significantly increased levels of RPE markers (MITF, TYR, TYRP2, PEDF, and BEST1) and the melanosomal protein PMEL17 were apparent when compared to cells differentiated in B27/N2 containing basal medium only.   However, LHX2 and ZO1 could be found in both pigmenting sheets and non-RPE cells at this time point.   Efficiency using PMEL17 as a marker was estimated at 78.5% compared to 25.2% for cells differentiated in basal medium and at this stage of differentiation (14 days), visible sheets were mechanically isolated, dissociated into single cells, and replated in an RPE medium (Ahmado et al) in the presence of the Rho-associated protein kinase (ROCK) inhibitor Y27632 in order to generate a more homogenous RPE population.   After enrichment and culture for another 30 days, cultures exhibited homogenous expression of MITF, OTX2, LHX2, ZO1, and PMEL17, while mature markers (BEST1 and RPE65) were more heterogeneous.   Polarized protein trafficking in these cells was also observed (Integrin αv was localized apically compared with OTX2 nuclear expression) and functionality was partly confirmed through the ability of hESC-RPEs to carry out phagocytosis (as measured by fluorescently labelled reactive oxygen species) at a higher level than foetal RPE.

These reports represent a step forward in the generation of RPE from hPSCs; a highly sought after cell type for clinical use.   The authors claim that speed of generation is increased, labour intensity is reduced, protocols are simplified, large scale production of cells is made amenable, growth medium requirements are reduced and the RPE cells produced are pure and deemed functional.   All these facets should make hESC-derived RPE cells amenable for clinical use for the treatment of age-related macular degeneration and other disorders.  


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From Stem Cells Translational Medicine.

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.