1.1. in culture, molecular analysis showed expression
1.1. Differentiation of humanembryonic stem cells into retinal pigment epithelial cells in feeder- andserum-free mediumWe have developed a new protocol for deriving RPE cells from Relicell®hES1 using direct differentiation method as shown in Fig.
1. Recently,it had been demonstrated that NIC promoted neural and subsequently RPEdifferentiation by preventing apoptosis of neuroectodermal cells (Idelson et al., 2009). Inthe first stage of differentiation, we added NIC and IGF-1 to initiatedifferentiation towards the neuroectoderm and the eye-field stage.
Post 28 daysin culture, molecular analysis showed expression of MiTF while there was noexpression of Rx seen in these cultures. These results suggested that ourcombination of growth factors induced RPE progenitors. We next changed theculture conditions by addition of APRE-19-conditioned medium for maturation ofthese progenitors to form RPE cells. ARPE-19 is a spontaneously arising humanRPE cell line with normal karyology and has structural and functionalproperties characteristic of RPE cells invivo.
When cultured in the presence of conditioned medium from ARPE-19cells, these RPE progenitors started forming RPE-like cell clusters in cultureby day 49. They appeared as pigmented cell clusters under phase-contrastmicroscope. Later, these cells accumulated more pigmentation and adopted atypical hexagonal morphology with a squamous appearance (Fig. 2A). Further,hESC-derived RPE cells formed prominent colonies of pigmented cells visiblewith the naked eye beyond 100 days in culture (Fig. 2B).
1.2. Gene expression andimmunofluorescence study of hESC-derived RPE cells We performed semi-quantitative RT-PCR for known markers at differentstages of differentiation (day 0, 14, 28, 60, and 100) in Relicell®hES1and compared their expression levels with adult human retinal RNA and ARPE-19(A-19) cell line (positive control), and UCMSCs (negative control). Our resultsdemonstrated that Relicell®hES1 expressed Oct-4 on day 0 and asdifferentiation was initiated there was a loss of Oct-4 expression (day 14, 28,60, and 100).
We also observed that bothneuroectodermal and early eye-field markers, Nestin and Pax6, were expressedthroughout the differentiation stages. Early neural-retina marker, Rx, showedvery faint expression by day 14 and disappeared thereafter. Expression of Chx10was seen by day 14 and up to day 28 that was later down-regulated as thesecells showed commitment towards RPE cell fate. The early RPE marker, MiTF, wasdetected as early as day 14 and the expression increased during differentiation(Fig.
3A). The RPE cell-specific markers, RPE65, CRALBP1, and Bestrophin, weredetected for the first time at day 60 and the expression of RPE65 increased byday 100 in Relicell®hES1. Other RPE cell markers, Otx2,PEDF, PMEL17, MERTK, and VEGF?A were expressed relatively early during thedifferentiation with TYRP1 being expressed by day 60.
The mesodermal marker,Brachyury, and the endodermal marker, GATA-4, were not expressed during any ofthe stages of differentiation. In addition, the early and late markers ofphotoreceptor differentiation, CRX, Recoverin, Opsin, and Rhodopsin, wereabsent in both these cells strongly suggesting the RPE cell fate of the derivedcells (Fig. 3B). Of the studied genes, undifferentiated cells showed theexpression of Nestin, Pax6, Otx2, PEDF, PMEL17, and VEGF-A; the undifferentiatedUCMSCs used as negative control also showed expression of PEDF and VEGF-A.
1.3. Electron microscopic analysisof hESC-derived RPE cellsFor any application of these derived cells, it is imperative thatthe cells possess functional capabilities.
Electron microscopic analysis ofhESC-derived RPE cells at day 100 (post-differentiation) was done to determinewhether the generated RPE cells have ultrastructural characteristics of theRPE. The cells were highly polarized with basally located nuclei; presence ofwell-developed tight-junction complexes; and apical microvilli (Fig. 4).Melanosomes responsible for pigmentation normally present in RPE cells wereclearly observed within the hESC-derived RPE cells.
Secretion of trophic factors viz.,VEGF-A and PEDF were analyzed by ELISA in cell supernatants collected at 48 htime interval. Results showed that hESC-derived RPE cells secreted significantlevels of VEGF-A and PEDF into the culture supernatant as compared to theundifferentiated hESCs (Data not shown). 1.
4. Expansion and growth of differentiated RPE cells We have shown that hESCs can be efficiently differentiated to formclusters of RPE that are tightly packed with cells. These cells show typicalhexagonal morphology and dense pigmentation in culture. One of the questionsaddressed was whether we can expand these cells to larger numbers suitable forregenerative cell therapy.
In the first attempts, we dissociated these RPEclusters to single cells with various methods (Supplementary information, TableS2). However, these techniques did not help as either the cell clusters did notdetach or there was a loss of RPE morphology. Since none of the methods yielded satisfactory results,we attempted to remove cells that were around RPE clusters. Using TrypLETMtreatment, we dissociated the cells surrounding the RPE clusters and removedthem from the dish. The RPE clusters were then maintained in culture for aperiod of 7 days in DMEM supplemented with B27 medium. There was no changeobserved in the morphology of RPE clusters. We treated theses clusters with reducedserum and cocktail of growth factors (PDGF-CC, rhEGF, bFGF) thereafter and itwas interesting to note that the cells started budding out of the clustersbetween days 4 and 7 and formed a monolayer of cells in culture by day 15.These RPE cells were further dissociated with TrypLETM and furtherexpanded and matured in culture multiple times.
1.5. Maturation of expandedhESC-derived RPE cellsSince we could now expand hESC-derived RPE cells, we investigated ifthese expanded cells could be made to regain hexagonal RPE morphology. HumanESC?derived RPE cells were first seeded on MatrigelTM and culturedfor 6-7 days in DBS-PEF medium. The cells expanded in these conditions but didnot show any signs of pigmentation or hexagonal morphology normally visible inRPE cells. It has been shown by various investigators that upon withdrawal ofbFGF, RPE cells can spontaneously differentiate into pigmented cells formingtypical RPE cell morphology (Klimanskaya et al., 2004).
Upon reachingconfluence, growth factors were withdrawn and the cells were further incubatedin DB medium. Within one week afterwithdrawal of growth factors, typical RPE monolayers with mild pigmentation wasobserved in culture (Fig. 5) indicating that it is possible to regain RPEmorphology and pigmentation in the expanded hESC-derived cells. At this time,they were designated as expanded and matured RPE (ExMat-RPE) cells. Further, hESC-derived RPE cells cultured onMatrigelTM developed heavy pigmentation by day 21 that was visibleto the naked eye, in contrast to those grown on tissue culture treated plastic surface(Fig.
6). 1.6. Gene expression analysis of ExMat-RPE cells ExMat-RPE cells were characterized to confirm RPE maturation. Expressionof specific markers associated with the cellular function of the mature RPE wasinvestigated by RT-PCR. Transcript expression levels of early neural marker,Nestin; eye-field marker, Pax6; early RPE marker, MiTF; mature-RPE markers,RPE65, CRALBP1, and Bestrophin; and melanogenic marker, TYRP1 were specificallydetected in ExMat-RPE cells. OtherRPE cell markers such as Otx2; neurotrophic factor, PEDF; phagocytic marker,MERTK; immature melanosome marker, PMEL17; and VEGF-A were also expressed inthese cells.
The pluripotent stem cell marker, Oct-4 was expressed in theundifferentiated hESCs and was not detected in ExMat-RPE cells. The mesodermal marker, Brachyury; the endodermalmarker, GATA-4; early neural-retinal progenitor marker, Chx10, was notexpressed in these cells. In addition, the early and late markers ofphotoreceptor differentiation, CRX, Recoverin, Opsin, and Rhodopsin, were alsoabsent in these cells as expected. Adult retinal RNA was used as positive control(Fig. 7). 1.
7. Characterization of ExMat-RPE cells byimmunofluorescence and flow cytometryImmunostaining of the ExMat-RPEshowed the expression of the early neural and eye-field markers, Nestin, Pax6,and MiTF; and mature-RPE markers,RPE65, CRALBP1, Bestrophin, Ezrin, CK18, and the tight-junction protein,ZO-1 (Fig. 8). Importantly, pigmented hESC-derived RPE cells did not expressthe pluripotent stem cell marker, Oct-4 but showed weak expression of SSEA-4indicating that the mature cells resembled adult RPE cells (Fig. 9). 1.8. Functional analysis of the ExMat-RPE cellsThe functionality of ExMat-RPEcells was shown by electron microscopy, phagocytosis, and polarized secretionof trophic factors.
Electron micrographs showed typical features characteristicof mature RPE that included pigmented cuboidal epithelial cells with apicalmicrovilli, abundant presence of melanin granules, and tight junctions betweenthe cells (Fig. 10). To simulate in vivoconditions in vitro, we used newlyintroduced pHrodoTM BioParticles® red conjugate to assessthe functional potential of these mature RPE cells. Immunofluorescencemicroscopy of cells immunostained with FITC-phalloidin showed theinternalization of fluorescently-labelled red pHrodoTM BioParticles®(Fig. 11).Polarized secretion of trophic factors was seen in ExMat-RPE cells. In this experiment, theculture supernatants were collected from the apical and the basolateral chamberof the porous Transwell insert (Supplementary information, Fig.
S1). The mean(± SEM) concentration of VEGF-A in the apical and basolateral supernatantswas 4.6 ± 0.08 ng/ml and 10.5 ± 0.09 ng/ml, respectively (Fig. 12A) while,the mean (± SEM) concentration of PEDF in the apical and basolateralsupernatants was 189.
5 ± 0.7 ng/ml and 196.5 ± 0.
5 ng/ml, respectively (Fig.12B). ExMat-RPE cells grown andmatured on Transwell culture membranes preferentially secreted VEGF-A to thebasolateral side while no significant difference in the secretion of PEDF wasnoted in these cultures. 1.9. Expression ofco-stimulatory molecules by ExMat-RPE cells in the absence and presence ofIFN-?In order to utilize hESC-derived RPE cells for allogeneictransplantation, it is very important to study the expression pattern ofco-stimulatory/ immunoregulatory molecules expressed by these cells. Weperformed gene expression studies on the ExMat-RPEcells that were untreated and treated with 10 ng/ml IFN-? for 5 days (Fig.
13A).ExMat-RPE cells showed B7-H1, B7-DC,IDO, HLA-G, and CD86 mRNA expression but no expression of HLA-DR and CD80 geneswere observed (Fig. 13B). However, mRNA expression corresponding to HLA-DRwas detected following IFN-? treatment for 5 days. IFN-? treatment of thesecells showed further increase in the mRNA levels of B7-H1, B7-DC, and IDO whilethere was no change in the mRNA expression of HLA-G and CD86.
The data obtainedfrom one representative experiment are shown in Fig. 13B. Next, immunophenotypic analysis for the expression ofco-stimulatory/ immunoregulatory proteins was also performed on ExMat-RPE cells. In comparison tocontrol cells, a significant increase in the expression of HLA-DR (58.3% ±2.3%), B7-H1 (78.
0% ± 3.1%), IDO (99.1% ± 2.9%), and HLA-ABC (84.1% 3.6%) wasnoted on day 5 after IFN?? treatment. As compared to ARPE-19 cells, IFN-? didnot induce expression of B7?DC, HLA-G, CD80, and CD86 molecules in ExMat-RPE cells. Representative resultsare shown in Fig.
13C. 1.10. Effect of ExMat-RPE cells on allogeneicT cell proliferation in vitroPurified allogeneic CD3+ T cells isolated from healthydonors were labelled with Carboxyfluorescein diacetate N-Succinimidyl Ester (CFSE)dye and co-cultured with ExMat-RPEcells in vitro for a period of 5 daysin a mixed lymphocyte reaction (MLR) study as previously described (Vasania et al., 2011).
Adecrease in the fluorescence intensity of CFSE was considered as an indicationof T cell proliferation. ExMat-RPEcells failed to elicit CD4+ T cell proliferation after 5 days of culture(Fig. 14). These cells retained maximum levels of CFSE dye (Fig. 14C) at anintensity comparable to that of T cells that were cultured alone (Fig. 14A)suggesting that the allogeneic T cells did not proliferate in the presence of ExMat-RPE cells during the 5 day cultureperiod.
Monocyte-derived dendritic cells (mDCs) were used as positive controland, as expected, mDCs induced 45.0% of CD4+ T cell proliferation inthe same assay (Fig. 14B). ExMat-RPEcells, interestingly, did not induce T cell proliferation even after exposingthe cells to IFN-? (Fig.
14D). These results indicate that ExMat-RPE cells are unable to induce allogeneic CD4+T cell proliferation in the presence of IFN?? and after upregulation ofHLA-DR antigen as shown earlier.