Пролиферация и пластичность клеток пигментного эпителия сетчатки

Пролиферация и пластичность клеток пигментного эпителия сетчатки: Comparison

Please note this is a comparison between Version 1 by Lyubov Rzhanova and Version 3 by Catherine Yang.

TheКлетки пигментного эпителия сетчатки (ПЭС) retina is a specialized light-sensitive tissue in the eye of mammals and humans that provides visual perception, and is actively studied at the cellular, molecular and genetic levels. Photoreceptor cells located in its outer part perform the function of converting light (phototransduction) into neurochemical signals, which are processed in the neurons of the retina and the brain and ultimately form our vision. Functional support for retinal neurons is provided by retinal pigment epithelium cells (RPE cells). The retinal pigment epithelium (RPE) is a single-row layer of pigmented, hexagonal, normally non-proliferating cells located between the choroid and the photoreceptor cells of the retina. The RPE performs many diverse functions to support the retina, including the transepithelial transport of substances, the phagocytosis of photoreceptor outer segments, and a number of processes in the visual cycle, as well as participation in the blood–retinal barrier and secretion of growth factors. The RPE plays an important role in regulating the redox homeostasis of retinal photoreceptors. A few cells have been isolated from the human RPE, which, according to strict clonal analysis and other stem cell criteria (self-renewal and the production of differential progeny), were classified as adult RPE stem cells (RPESCs). The number of mammalian RPESCs was determined in vitro experiments, from which it became clear that to 2% of cells are capable of proliferation, self-renewal, and the expression of specific genes characterizing stem cells. Depending on microenvironmental conditions, RPESCs can remain quiescent in a stemness state or exhibit multipotent differentiation. RPESCs can produce RPE cells and are capable of generating different types of photoreceptors and nerve cells, or mesenchymal cellsимеют фундаментальное значение для развития и функционирования сетчатки. Сетчатка — специализированная светочувствительная ткань глаза млекопитающих и человека, обеспечивающая зрительное восприятие, активно изучаемая на клеточном, молекулярном и генетическом уровнях. Клетки фоторецепторов, расположенные в его внешней части, выполняют функцию преобразования света (фототрансдукции) в нейрохимические сигналы, которые обрабатываются в нейронах сетчатки и головного мозга и в конечном итоге формируют наше зрение. Функциональную поддержку нейронов сетчатки обеспечивают клетки пигментного эпителия сетчатки (клетки RPE). Пигментный эпителий сетчатки (ПЭС) представляет собой однорядный слой пигментированных шестиугольных, в норме непролиферирующих клеток, расположенный между сосудистой оболочкой и фоторецепторными клетками сетчатки. РПЭ выполняет множество разнообразных функций по поддержанию сетчатки, включая трансэпителиальный транспорт веществ, фагоцитоз наружных сегментов фоторецепторов и ряд процессов зрительного цикла, а также участие в работе гемато-ретинального барьера и секреции факторов роста. . РПЭ играет важную роль в регуляции окислительно-восстановительного гомеостаза фоторецепторов сетчатки.

retinal pigment epithelium
RPE
stem cells
RPESCs
differentiation
proliferation
plasticity
morphometry map
heterogeneity
cell subpopulation

1. ProliferationПролиферация of Retinal Pigment Epithelium Cellsклеток пигментного эпителия сетчатки

OnceКак только дифференцировка клеток РПЭ theзавершена RPE[ cell82 differentiation, is83 complete^[1]], theклетки выходят cells exit the cell cycle and persist throughout life, dividing rarely or not at all. Mammalian and human RPE cells are thought to be terminally differentiated, postmitoticиз клеточного цикла и сохраняются на протяжении всей жизни, делясь редко или не делясь вообще. Считается, что клетки РПЭ млекопитающих и человека представляют собой терминально дифференцированные постмитотические неделящиеся клетки. Однако есть данные, что у грызунов клетки РПЭ на периферии слоя способны к пролиферации [ 57 , nondividing84 cells]. However, there is evidence thatИзвестно также, что периферические клетки РПЭ макак могут включать 3Н-тимидин, что указывает на их пролиферацию [ in28 rodents, the85 RPE]. cells at the periphery of the layer are capable of proliferationЭта информация дополняется данными о дифференциальной экспрессии генов, связанных с клеточным ^[2]циклом. ItТак, было показано, что в периферических клетках is also known that the peripheralРПЭ доминируют гены, стимулирующие пролиферацию [ RPE57 cells].

Помимо ofпроявления macaques can incorporate 3H-thymidine, indicating their proliferationпролиферативной активности in situ, клетки РПЭ взрослых млекопитающих и человека могут активироваться к делению при ^[3].помещении Thisв information is complemented by data on the differential expression of genes associated with the cell cycle. Thus, it was shown that genes stimulating proliferationкультуру клеток, где их пролиферация облегчается потерей межклеточных контактов и различными факторами роста в культуральной среде. Предполагается, что пролиферация и амплификация клеток РПЭ in vitro происходит преимущественно за счет субпопуляции стволовых клеток (СК) в РПЭ [ 86 ].

2. Экспрессия маркеров стволовых клеток и потенциал дифференцировки клеток РПЭ

Экспрессия маркеров мультипотентности и стволовых клеток в РПЭ является важным критерием, позволяющим отнести dominatedнекоторые in peripheral RPE cellsклетки РПЭ к взрослым ^[4]СК. InОднако для того, чтобы клетки можно было отнести addition to exhibiting proliferative activity in situ, adultк СК, они должны обладать рядом характеристик, таких как самообновление и генерация специализированного дифференцированного потомства. Самообновление — это способность СК пролиферировать симметрично или асимметрично и продуцировать аналогичные клетки [ 87 ]. Замена поврежденных или потерянных клеток во mammalianвремя and human RPE cells can be activated to divide when placedрегенерации происходит посредством различных механизмов, включая дедифференцировку, трансдифференцировку или репрограммирование [ into88 a, cell89 culture, 90 ]. whereВсе три механизма можно наблюдать в слое РПЭ глаза позвоночных. Классические эксперименты на модельных животных показали способность their proliferation is facilitated by the loss of intercellular contacts and various growth factors in the culture medium. Inклеток РПЭ трансдифференцироваться/перепрограммироваться в нервные клетки сетчатки, что успешно воспроизводится in vivo у низших позвоночных. Дедифференцировка — это переход окончательно дифференцированных клеток в менее дифференцированное состояние, в котором клетки могут делиться и в пределах своей клеточной линии локально заменять утраченные клетки, переходя к их окончательному дифференцированному состоянию у некоторых хвостатых studiesамфибий of[ the89 mechanism]. of entry of human RPE cells into the phase of DNA synthesis in vitro, it was found that mitogen-activated proteinВо время трансдифференцировки клеток RPE после повреждения сетчатки у этих животных уже дифференцированные клетки RPE меняют клон и перепрограммируются в нейрогенные предшественники, экспрессирующие Klf4, Sox2, Pax6 и c-Myc [ kinase89 (MAPK)]. and extracellular signal-regulated kinase (ERK) play a key role. In this signaling pathway, growth factorПотомки этих клеток-предшественников дифференцируются во все клетки сетчатки, включая фоторецепторы, глию и пигментный эпителий, а затем полностью восстанавливают функцию сетчатки у амфибий [ receptor89 activates, Ras91 GTPase, leading92 to, MAPK/ERK93 phosphorylation]. The MAPK/ERK signaling pathway, inПовреждение сетчатки у млекопитающих и человека приводит к развитию патологий, сопровождающихся приобретением клетками РПЭ мезенхимального фенотипа [ 89 turn, 93 ]. Утрата межклеточных regulatesконтактов запускает механизмы ЭМП. После накопления необходимого пула клеток-предшественников начинается ЭМП и дифференцировка в разные типы клеток. Некоторые исследователи проводят параллель между этими процессами в сетчатке и transcription factors,эмбриональным развитием, а также развитием c-myc, Pax6, klf4,опухолей [ 88 ]. andВ MITF,связи theс expression of which indicates a decrease in the level of RPE differentiationэтим у взрослых млекопитающих и человека трансдифференцировка РПЭ как средство регенерации сетчатки ^[5]малоэффективна. TheОднако некоторые ability to proliferate has been shown to be maintained through the persistent repression of cyclin-dependent kinase inhibitorклетки РПЭ у млекопитающих могут проявлять пролиферацию, пластичность и преобразование в нейроны, что можно обнаружить in vitro [ (CDKI)28 genes, including57 p16, Arf, p21, and84 p57, wh85].]. При репрограммировании ichn were expressed at very low levels in the RPE stem cells (RPESCs), compared with their parental RPE cellsvitro полностью дифференцированная клетка может частично вернуться в свою плюрипотентную стадию, приобретя лишь некоторые характеристики СК, затем пролиферировать и дифференцироваться в другие типы ^[6]клеток. ItУ птиц и is assumed that the proliferation of RPE cells in vitro occurs mainly due to a subpopulation of stem cells (SCs) in theмлекопитающих трансдифференцировка РПЭ в нейроны сетчатки происходит во время раннего эмбрионального развития только под влиянием основного фактора роста фибробластов (bFGF) или lin-28 [ RPE94 ^[5]].

23. Expression of Stem Cell Markers and Differentiation Potential of the RPE Cells Дифференцировка стволовых клеток/клеток-предшественников пигментного эпителия сетчатки в мышечные, адипо-, остео- и хондрогенные клетки.

TheВ ряде исследований из РПЭ человека было выделено несколько клеток, которые согласно строгому клональному анализу и другим критериям стволовых клеток (самообновление и производство дифференциального потомства) были классифицированы как стволовые клетки взрослого replacement of damaged or lostРПЭ: РПЭСК. -производный RPE (RPESCs-RPE) [ 95 , 96 , 97 , 98 , 99 , 100 ]. Термин cellRPESCs during regeneration occurs through-RPE был впервые предложен после того, как была показана способность RPESCs-RPE к мультипотентной дифференцировке в нервном и мезенхимальном (остеогенном, адипогенном и хондрогенном) направлениях [ various100 mechanisms].

Показано, что взрослый РПЭ including dedifferentiation, transdifferentiation, or vitro может генерировать клетки, экспрессирующие маркеры клеток мезенхимального происхождения: мышечные, адипо-, остео- и хондрогенные клетки [ reprogramming100 ^[7]]. Чтобы All three mechanisms can be observed in the RPE layer of the vertebrate eye. Classic experiments on model animals have shown the ability of RPE cells to transdifferentiate/undergo reprogramming into neural cells of the retina, which is successfully reproduced in vivo in lower vertebrates. Dedifferentiation is the transition of terminally differentiated cellsисключить контаминацию и доказать мультипотентность стволовых клеток, авторы получили RPESC из первичного RPE человека путем клонирования одиночных клеток. Клетки помещали в разные культуральные лунки и подвергали воздействию среды для дифференцировки адипоцитов, хондроцитов и костей. Это исследование было проведено на клетках из различных источников, включая фетальный РПЭ человека, РПЭ различных видов млекопитающих, клетки ARPE-19 и клетки РПЭ, полученные из индуцированных плюрипотентных клеток и СК. Результаты данного исследования показали, что для РПЭ характерна мезенхимальная дифференцировка. Было обнаружено, что РПЭ плода человека наиболее устойчив к приобретению мезенхимальных судеб. В недавних исследованиях были изучены стволовые клетки фетального РПЭ (fRPESC), полученные под воздействием витамина С и вальпроевой кислоты [ to27 a]. less differentiated state in which cells can divide and, within their cell lineage, locally replace lost cells, moving to their final differentiated state in some tailed amphibiaКлетки fRPE и fRPESC были помещены в адипогенную, хондрогенную и остеогенную среду дифференцировки, чтобы проверить их способность к мезенхимальной дифференцировке. Окрашивание клеток специфическими маркерами и количественная ПЦР в реальном времени показали, что fRPESC более способны к дифференцировке в адипоциты, хондроциты и в ^[8].остеогенном Duringнаправлении, the transdifferentiation of RPE cells after retinal damageчем клетки fRPES. Была выявлена регуляторная роль inSOX2 theseв animals, the already-differentiated RPE cells change lineage and reprogram into neurogenic progenitors expressingклеточной конверсии fRPESC и показано, что fRPESC теряют способность к мезенхимальной дифференцировке после нокдауна Klf4, SoxOX2, Pax6, and[ c-Myc27 ^[8]]. TheНаряду с descendants of these progenitor cells differentiate into all retinal cells, including photoreceptors, glia, and pigment epithelium, and then fullyдифференцировкой РПЭСК в мезенхимальном направлении in vitro, из многолетних патоморфологических наблюдений хорошо известно, что в глазах человека иногда обнаруживаются хрящевые и костные образования, развивающиеся из клеток РПЭ [ restore100 retinal, function101 in, amphibia102 ^[8]]. InЭти данные birds and mammals, RPE transdifferentiation into retinal neurons occursуказывают на то, что RPESCs имеют широкий репертуар дифференцировок, напоминающий таковой у клеток нервного гребня [ during103 early]. embryonic development, only under the influence of basic fibroblast growth factor (bFGF) or lin-28Можно предположить, что эта мультипотентность RPESCs может быть каким-то образом связана с очень ранним высвобождением нейроэпителиальных клеток-предшественников RPE во время ^[9]эмбриогенеза.

Сведения Damageо to the retina in adult mammals and humans leads to the development of pathologies accompanied by the acquisition of a mesenchymal phenotype by RPE cells, which begin to actively proliferate andдифференцировке РПЭСК в нейральном направлении в глазах млекопитающих и человека in vivo отсутствуют, тогда как развитие в мезенхимальном направлении с образованием эпиретинальных мембран при патологиях сетчатки является общеизвестным медицинским фактом [ migrate100 ^[10]., The104 loss]. .

4. Дифференциация стволовых клеток/клеток-предшественников из пигментного эпителия сетчатки вдоль нейронального пути

Mof intercellular contacts triggers the mechanisms of epithelial–mesenchymal transition (EMT). After the accumue than 20 years ago, the researchers' laboratory established that in the total population of the necessary pool of altered RPE cells, these cells begin differentiateRPE cells of the adult human eye, the pluripotency genes iOCT4, NANOG, antod SOX2 various types of cells, predominantly of mesenchymal origin.e expressed, as well as the neural differentiation gene SomePAX6, researwhichers draw a parallel between these processes was identified in the retina and RPE during embryonic development, as well as the development of tumors ^[7]. In thindis regcard, in adult mammals and humans, RPE transdifferentiation as a means of retinal regeneration is ineffective. However, some RPE ting that a certain proportion of cells in mammals may exhibit proliferation, plasticity, and conversion to neurons, detectable in vitro .

Thwith stem/progenitor properties are expression of multipotency markers and stem cells ossibly present in the RPE i[105,106,107]. Subs aequen important criterion that allows us to classify somet studies of the behavior of adult and fetal RPE cells as adult SCs. The somein vitro showed that cells of native human RPE and cell cin culture have been characterized by the ededifferentiate and expression of stem stem cell and poorly differentiated cell markers OCT4, including NANOG, OCT4, SOX2, SSEPA-4X6, KLF4, C-MYC, and LIN-28 ^[11][12]PROX1. HowUndever, in order for cells to be classified as SCs, they must possess a number of characteristics,r in vitro differentiation conditions, the cells express neural and glial cell markers such as sNESTIN, MSI1, βIII-Tubulin, neurofilf-renewal and the generation of specialized differentiated progeny. Self-renewal is the ability of SCs to proliferate symmetrically or asymmetrically and to produce a similar cellaments (68–200 kDa), and GFAP. In addition, using immunostaining, markers of mature retinal and brain cells ^[13]. Thwe proliferation of human RPESCs was stimulated by cultivating them in a free-floating state with the addition of growth factors, similar to neural stem cells (NSCs)e detected: recoverin (photoreceptor marker), ^[11][12]. Cldonpal analysis of adult RPE cells was used and it was shown that primary spheres from free-floating cells were formed after 4 days with a frequency of 1.5% of the number of initial seeded cellsminergic neuron markers, tyrosine hydroxylase (TH) (retinal amacrine cell ^[12]. Thmarker), RPE cells were cloned in adherent cultures to expand the number of RPESCs ^[14]GABAergic interneuron markers, using protocols for isolating and culturing RPESCs from previous studies ^[15][16]. In the NOS (retinal amacrine or gadherent cultures, about 10.6% of the cells actively proliferated, although the vast majority did not divide or produced only limited progeny ^[12]. Iglion cell marker), as well as CNPase and O4 (oligodendrocyte was found that 10% of RPE cells proliferate once in culture, using tmarkers).

The clonal plating. Among RPE cells, only 2% of cells proliferate very actively and can create up to 90% oftive human RPE and cell culture have been characterized by the entire monolayer. These cells were classifiedxpression of stem cell markers, including NANOG, asOCT4, adultSOX2, RPESCsSSEA-4, ^[14].KLF4, TheC-MYC, humand fetaLIN-28. Cul RPE (fRPE) was treated with vitamin C and valproic acid; the stemness properties of the resulting fetal RPESCs (fRPESCs) were studied, and a significant increase in the stem cell markers SOX2, OCT4ured RPE cells were positive for the surface marker SSEA4, showed little reaction to SOX2 immunostaining, and KLF4 showased found ^[17]. Ano immunos ta result, the cells enter a retinal stem cell-like stateining for OCT4 and NANOG [100]. It was found that the combined effect of vitamin C and valproic acid activates the expression of the retinal progenitor markers MITF, OTX2, and PAX6, as well as the mesenchymal stromal markers CD133, CD73, CD105, and CD90 in fRPE cells. Researchers believe that a high expression of SOX2 in fRPESCs is a prerequisite for maintainingAs a result, the cells enter a retinal stem cell properties and a multipotent differentiation potential-like state ^[17]. In another study, (fRPESCs). were activated by the influence of amniotic fluid factors, and the retinal progenitor cells obtained in this way were studied ^[18]. The mResearchers believe that a high expressiouse RPE cells were cultivated in the form of spheres to obtain induced iof SOX2 in fRPESCs. If mouse RPE cells are cultured through the sphere formation stage, approximately 0.003–0.013% of RPESCs can be activated is a prerequisite for maintaining ^[6]. Threse cells actively proliferated for more than ten passages, in contrast to other RPE cells, which showed limited proliferation and senescence after only five passages. From the above studies, it follows that in the adult human and mouse RPE cells are preserved that have signs of SCs that can be identified in vitronal stem cell properties and a multipotent differentiation potential [27].

The search for and study of the stem properties of the RPE continues, using modern molecular genetic technologies. The RNA-Seq and scRNA-Seq studies of the functional heterogeneity of the native RPE cells of mice and humans were found subpopulations of cells expressing stem/progenitor cell genes ^[19][20]. Among tAmong the identified mouse RPE clusters, researchers paid special attention to cluster C1, containing only 1–2% of the total number of cells ^[21][67], and cluster 5, in which only 19 cells (0.59%) were found ^[22][26], which were cells with the characteristics of stem cells and/or progenitor cells. In these clusters, a high expression of stemness and stem cell maintenance genes was observed, against the background of a high expression of melanogenesis genes. This indicated that the cells were still in the process of differentiation. It was also found that there was a high correlation of gene expression between RPE cells of the C1 cluster and retinal cells, which possibly pointed to the ability of C1 cells to differentiate into different cell types of the neural retina ^[21][67]. In addition, it was demonstrated that the RPE cells in the adult mouse eye are epigenetically very similar to the phenotypes of retinal progenitor cells and photoreceptors ^[23][111]. Several growth factors, such as Vegf, Tgf, Pdgf, Egf, and Ngf; their receptors, Vegfr, Egfr, Pdgfr, and Tlk4; and two stem cell-associated signaling ligands, Kit-l and Lif, are involved in mouse RPE reprogramming into induced RPE stem cells (iRPESCs) and are highly expressed in iRPESCs.

Mouse RPESCs were induced in vitro through the stage of sphere formation (sphere-induced RPE stem cells, iRPESCs) and it was discovered that the gene expression profile of iRPESCs was very different from the parental RPE. At the same time, changes in gene expression occurred immediately after the formation of spheres. Mouse iRPESCs expressed increased levels of c-Myc, Oct4, c-Kit, and Cd44 ^[6] [96]. The expression of eight of the fifteen selected stemness genes, such as Klf4, Alpl, Kit, Kitl, and Bmi1, was significantly upregulated in RPE spheres, and the expression of Abcg2, Bmi1, Cd44, Kitl, and c-Myc was upregulated in iRPESCs, compared with the original RPE. Two DNA methylation genes (Dnmt1 and Dnmt3a), four histone acetylation genes (Hat1, P300, Myst2, and Myst3), and seven deacetylation genes (Sirt2, Sirt6, Hdac1, Hdac2, Hdac3, Hdac5, and Hdac6) were highly expressed in the stem cells, reflecting epigenetic regulation that may promote the expression of major stemness genes, such as Oct4 and Klf4 in iRPESCs ^[6][96,112]. From t

The abprove studies, it follows that among the heterogeneous RPE cells of mice and humans, 0.003–2% of cells can be distinguished that are capable of proliferation, self-maintenance, and the expressionliferation of human RPESCs was stimulated by cultivating them in a free-floating state with the addition of growth factors, similar to neural stem cells (NSCs) [100,105,106,110]. The mof specific genes characterizing the state of stemness in cellsuse RPE cells were cultivated in the form of spheres to obtain induced iRPESCs [96]. These RPE cells may remain quiescent or exhibit multipotent differentiation into a mature cell type. Under expanwere cloned in adherent cultures to expand the number of RPESCs [98], usion culture conditions in whichg protocols for isolating and culturing RPESCs self-renewfrom previous studies [95,97,99,113,114]. tThey generate SC progeny, and, conversely, under differentiation culture conditions, human fetal RPE was treated with vitamin C and valproic acid; the stemness properties of the resulting fetal RPESCs (fRPESCs generate progeny of different cell types.

3. Differentiation of Stem/Progenitor Cells from the Retinal Pigment Epithelium into Muscle and Adipo-, Osteo-, and Chondrogenic Cells

Th) were studied, and a significant increase in the stem cell term RPESCsmarkers SOX2, OCT4, and KLF4 was first proposed ound [27]. In afnoter the ability of her study, RPESCs for multipotent differentiation was shown in the neural and mesenchymal (osteogenic, adipogenic, and chondrogenic) diwere activated by the influence of amniotic fluid factors, and the retinal progenitor cells obtained in this way were studied [115]. Frectionsm ^[12] . Ithe was demonstratedabove studies, it follows that an in the adult RPE in vitro can generatehuman and mouse RPE, cells expressing markers of cells of mesenchymal origin: muscle and adipo-, osteare preserved that have signs of SCs that can be identified in vitro.

Clo-,nal and chondrogenicalysis of adult RPE cells ^[12].was To usexclude contamination and to prove the multipotency of the stem cells, the authors obtained RPESCs from a primary human RPE by cloning single cells. Thed and it was shown that primary spheres from free-floating cells were formed after 4 days with a frequency of 1.5% of the number of initial seeded cells were[100]. placedIn into diffthe adherent culture ws, about 10.6% of the cells and exposed to adipocyte, chondrocyte, and bone differentiation mediactively proliferated, although the vast majority did not divide or produced only limited progeny [100]. This sItudy was conducted on was found that 10% of RPE cells from a variety of sources, including human fetal RPE, RPE from various mammalian species, ARPE-19proliferate once in culture, using the clonal plating. Among RPE cells, and RPEonly 2% of cells derived from induced pluripotent proliferate very actively and can create up to 90% of the entire monolayer. These cells and SCswere classified as RPESCs [98]. If mouse The results of this study showed that mesenchymal differentiation is characteristic of the RPE. It was found that human fetal RPE is most resistant to acquiring mesenchymal fatesRPE cells are cultured through the sphere formation stage, approximately 0.003–0.013% of RPESCs can be activated. These cells actively proliferated for more than ten passages, in contrast to other RPE cells, which showed limited proliferation and senescence after only five passages [27].

In rstudiecent research, stems of the mechanism of entry of human RPE cells from the fetal RPE were studiedinto the phase of DNA synthesis in ^[17].vitro, Iit was found that the combined effect of vitamin C and valproic acid activates the expression of the retinal progenitor markers MITF, OTX2, and PAX6, as well as the mesenchymal stromal markers CD133, CD73, CD105, and CD90 in fRPE cells. Cells of the fRPE and fRPESCs were placed into adipogenic, chondrogenicmitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) play a key role. In this signaling pathway, growth factor receptor activates Ras GTPase, leading to MAPK/ERK phosphorylation. The MAPK/ERK signaling pathway, in turn, regulates transcription factors, c-myc, Pax6, klf4, and osteogenic differentiation media to test their mesenchymal MITF, the expression of which indicates a decrease in the level of RPE differentiation capabilities[86,116]. Steveraining cells with specific markers and quantitative real-time PCR showed that fRPESCs are more capable of differentiating into adipocytes, chondrocytes, and in the osteogenic direction than fRPE cells. The regulatory role of SOX2 l growth factors, such as Vegf, Tgf, Pdgf, Egf, and Ngf; their receptors, Vegfr, Egfr, Pdgfr, and Tlk4; and two stem cell-associated signaling ligands, Kit-l and Lif, are involved in RPE reprogramming into in the ducellular conversion of fd iRPESCs was identified and it was shown that fand are highly expressed in iRPESCs lose t. The ability for mesenchymal differentiation after the knockdown ofto proliferate has been shown to SOX2 ^[17]be .maintained Along witthrough the differentiatpersistent repression of RPESCs in the mesenchymal direction in vitro, it is well known from many years of pathomorphological observations that the cartilagincyclin-dependent kinase inhibitor (CDKI) genes, including p16, Arf, p21, and p57, which were expressed at very low levels in the RPESCs, compared with their parental RPE cells [96].

Frousm the and bone formations that develop frombove studies, it follows that among the heterogeneous RPE cells are sometimes found in human eyesof mice and humans, ^[24]0.003–2% These data indicateof cells can be distinguished that RPESCs have a broad repertoire of differentiations, reminiscent of that of neural crestare capable of proliferation, self-maintenance, and the expression of specific genes characterizing the state of stemness in cells. These cells ^[25].may It creman be speculated that thisin quiescent or exhibit multipotency of RPESCs may be somehow related to the very early release of neuroepithelial RPEt differentiation into a mature cell type. Under expansion culture conditions in which RPESCs self-renew, they generate SC progenitor cells during embryogenesis. They, and, conversely, under differentiation culture condis no information about the tions, RPESCs generate progeny of differentiation of cell types. RPESCs in the neural direction in mammalian and human eyes in vivo, while development in thecan produce RPE cells, and epithelial monolayers of RPE cells are easily obtained. In in vitro differentiated media that activate neural or mesenchymal direcfferentiation, with the formation of epiretinal membranes in retinal pathologies, is a well-known medical factRPESCs can generate both neuronal and mesenchymal cells [86,100].

45. Differentiation of Stem/Progenitor Cells from the Retinal Pigment Epithelium along the Neuronal Pathway and into Retinal Photoreceptor Cells

SubUsequeint studies of the behavior of adult and fetal RPE cells in vitro showed that cells in culture dedifferentiate and express stem cell and poorly differentiated cell markers OCT4, NANOG, SOX2, PAX6, ag qPCR analysis and immunostaining, it was shown that und PROX1. Under in vitro differentiation conditions, the RPE cells express neural and glial cell markers such as NES, MSI1, TUBB3, NF-L/H, GFAP. Iof embryonic and addition, using immunostaining, markers of mature retinal and brain cells were detected: RCVRN (photoreceptorult human eyes express marker), dopaminergic neuron markers, tyrosine hydroxylase (TH) (retinal amacrine cell marker), GABAergic interneuron markers, nNOS (retinal amacrine or ganglion cell marker)s of progenitor and neural cells [105,106,108,109,110]. as well as CNPase and O4 (oligodendrocyte markers) ^[26]. Otther researchers have found photoreceptor markers in RPE cell lines and primary RPE cultures, such as Crx10 [117], Opn3, ^[27]Opn4, Nrl, Crx, Opn1mw/1w, OPN3, OPN4Sag, NRL, CRXNr2e3, OPN1MW/1W SAG, NR2E3,and RCVRNrecoverin ^[28][118]. The identification of the recoverin protein suggests that human RPE cells are capable of differentiating into retinal photoreceptor-like cells. In that study, the human RPE cells were cultured in a medium that promotes the differentiation of retinal neurons ^[12][100]. Using qPCR analysis, it was shown that the expression of the neural progenitor marker NES and the neuronal marker TUBB3 was significantly activated in RPE cells (over 1000-fold and 90-fold, respectively). Interestingly, under these conditions, the cells increased the expression of the eye field marker genes LHX2, OTX2, and RX, which are characteristic of the early stages of eye development, while the levels of retinal progenitor markers CHX10 and RHO did not change, and the expression of PAX6 actually decreased. These culture conditions have been noted to promote the differentiation of RPESCs toward neural progenitors of the forebrain and retina ^[12][100].

The directed differentiation of human fRPESCs into photoreceptors using a special three-step protocol using different culture systems and media has been carried out ^[17][27]. During the process of differentiation, the fRPESCs changed their morphology, first forming a round shape and then extending several synapse-like structures to finally form a tubular rod-shaped structure resembling the outer segment of a photoreceptor. At the first stage of differentiation, retinal photoreceptor progenitor cells with the expression of markers PAX6 and VSX2 were obtained. At the second stage, the cells were differentiated into photoreceptor progenitors that significantly increased the expression of photoreceptor markers NRL and CRX. Finally, at the terminal stage of RPESC differentiation, rod photoreceptor cells expressing REC, RHO, ARRESTIN, and GNAT1 were obtained ^[17][27]. Another study managed to differentiate fetal RPE cells into rod photoreceptors by chemical reprogramming ^[29][14]. Gene ontology analysis of RNA sequencing results from these cells revealed the upregulation of genes involved in neuronal generation, neurotransmitter uptake, and photoreceptor cell differentiation, such as SOX8, IGFN1, ASCL1, RXRG, THRB, and RORB. On day 10 of reprogramming, the transcriptome profile showed a stable activation of genes specific to rod photoreceptors, but not to cones ^[29][14].

56. Differentiation of Stem/Progenitor Cells from the Retinal Pigment Epithelium along the RPE Pathway (Redifferentiation)

Mouse and human RPE stem cells are capable of redifferentiating to the original phenotype. The sphere-derived dedifferentiated RPESC-like cells can differentiate back into RPE cells in vitro ^[17][27,116]. Monolayer cultures of RPE cells derived from human RPESCs (RPESC-RPE) have been described and characterized as cultures with the morphology and physiology characteristic of the native RPE ^[15][16][95,97,99,113,114]. The redifferentiation of RPESCs occurs within 8 weeks of cultivation. During this time, the cells change their morphology, proliferation rate, and polarization and also acquire the key phenotypic characteristics of the RPE. In cultured spindle-shaped RPESCs, a significant decrease in proliferation was observed after 2 weeks; after 4 weeks, the appearance of islets of cells with a cuboidal morphology was noted, and, by week 8, almost all cells acquired the morphology of a mature RPE. The secretion of vascular endothelial growth factor A (VEGF-A) through the apical and basement membranes increased from week 2 to week 4, while the secretion of pigment epithelium-derived factor (PEDF) appeared during week 6 of the cell differentiation^[16] [95,97,99]. In the cultured adult RPE, a similar but sometimes increased expression of RPE markers was observed, compared with the native tissue. Claudin-19 was present along the apical–lateral membrane along with the tight junction protein ZO-1, indicating the existence of a functional epithelial barrier. Ezrin, a membrane-associated protein involved in cytoskeletal organization, was found predominantly in the RPE microvilli. The visual cycle proteins, cellular retinaldehyde-binding protein (CRALBP) and RPE65, were localized, as expected, in the cytoplasm. Monocarboxylate transporter 1 (MCT1) was present on the apical surface of cells ^[14][98]. This indicates the establishment of the polarity characteristic of mature RPE cells. It is obvious that, in mouse and human RPEs, cells with stem properties are present, which also have the ability to redifferentiate to the original phenotype of RPE cells.

Throughout the entire period of differentiation, the RPESC-RPE cells continuously expressed pigment epithelium cell fate determination factors OTX2 and MITF and did not express smooth muscle actin, an indicator of epithelial–mesenchymal transition. It has been shown that, during the differentiation period, transepithelial resistance increases and reaches the norm ^[15][95,99]. However, the phagocytic activity of RPESC-RPE cells decreased after 4 weeks of differentiation, apparently due to a lack of contact with photoreceptors, which are necessary for the full maturation of the RPE ^[16][97].

RNA-Seq and scRNA-Seq on differentiated RPE cells derived in vitro from human RPESCs identified 13 cell subpopulations with different functional specializations. The transcriptomic analysis showed that the subpopulation composition of the entire RPE cell population is subject to dynamics over time. These findings suggest that RPE subpopulations have overlapping but distinct functional profiles. Among these subpopulations, othere are tene subpopulation stands out in which a profiles in number. In these cells, the specific marker EZH2 (a transcription factor involved in histone methylation, self-renewal, and the differentiation of SCs), has been was isolated, indicating a stemate similar to stem cells or progenitor cell-like states [71].^[30]. This study has confirmed RPE heterogeneity under culture conditions, similar to the native tissue, and answered the previously raised question about the innate differences between RPE cells, and whether a trait can be accurately passed on to daughter cells in the reproducing population [31]. It is likely that the mosaicism of RPE cells is hereditary in nature, embedded in the genetic program of the cells themselves, and conditioned by epigenetic influence. Based on these data, the human RPE may contain highly plastic cells that have (or acquire during the process of dedifferentiation) the SC phenotype with a dual potential. These cells may retain the potential to redifferentiate into RPE and to differentiate into retinal cells, under the influence of appropriate microenvironmental factors. RPESCs can produce RPE cells, and epithelial monolayers of RPE cells are easily obtained. In in vitro differentiated media that activate neural or mesenchymal differentiation, RPESCs can generate both neuronal and mesenchymal cells ^[12].

6. The Niche of Stem/Progenitor Cells from the Retinal Pigment Epithelium and Localization in Tissue RPE

It is obvious that RPESCs are not primitive cells but are differentiated cells, morphologically indistinguishable from the neighboring cells. The stem state of RPESCs maintains a well-structured niche microenvironment consisting of Bruch’s basement membrane, intercellular lateral contacts in the RPE, and apical contacts with the outer segments of photoreceptor cells. Cell proliferation is controlled by the niche components in interaction with endogenous signals ^[32]. Like neural stem cells in the brain ^[33], RPE stem cells are quiescent and require changes in the microenvironment and signals in the niche for their activation and proliferation. The factors that activate the reprogramming of RPE cells and dormant stem cells are the same. This means the loss of intercellular contacts and contacts with surrounding tissues, the remodeling of the cell surface and cytoskeleton, the displacement of RPE cells from their stabilizing environment (niche), proliferation, migration, genome reprogramming, and, ultimately, the sequential acquisition of a different phenotype^[34].

The RPE maintains tissue homeostasis through endogenous regulatory systems. Mature RPE can be compared with classical barrier epithelia, which are characterized by a constant cellular turnover throughout life, such as the corneal epithelium, skin epidermis, or intestinal epithelium ^[32]. To ensure the maintenance of tissue homeostasis, the rate of cell removal must match the production of new cells, which is achieved primarily through stem and progenitor cells. Mature RPE maintains tissue homeostasis mainly through long-term cell survival, although cell density decreases with age, especially in the peripheral RPE. The decrease in cell density is thought to be due to the continued growth of the eye during the juvenile period, and the gradual loss of senescent cells ^[35]. Maintaining the integrity of the RPE is achieved through hypertrophy without loss of contact with neighboring cells and/or through cellular replacement (migration from the periphery), which may explain the relative preservation of the middle part of the RPE, and a sharp decrease in the cell density in the periphery ^[36]. The long life of RPE cells does not exclude the possibility of the presence of a few stem/progenitor cells among them, although the question of their localization remains open. It is assumed that less differentiated RPE cells retain the ability to transdifferentiate into other cell types ^[32]. RPESCs should be located in a topographic zone with less differentiated cells, where there are less rigid cell contacts; this is the peripheral RPE ^[4], which is as close as possible to the ciliary marginal zone. From an evolutionary point of view, it would seem that RPESCs can be preserved in the ciliary marginal zone of the eye. It was also suggested that the RPE subpopulation of the immediate periphery (zone P4) may contain a pool of RPE cells that retain the ability to proliferate ^[37]. In contrast to these assumptions, the scRNA-Seq results clearly showed that less differentiated cells are found in the central area of the RPE, in particular, in the macular region ^[19]. In the cells of this area, a high expression of stemness and stem cell maintenance genes was found, against the background of a high expression of melanogenesis genes, which indicates an ongoing process of cell development.

7. The Potential of Retinal Pigment Epithelium Cell Subpopulations for Transplantation

Retinal degeneration as a result of the death of photoreceptors and RPE is the main cause of many degenerative diseases of the human eye, leading to vision loss ^[38][119]. For vision correction during this kind of pathology, approaches are currently being actively developed in contemporary medicine, aimed at preserving the original photoreceptors and RPE ^[39][120] and/or striving to replace cells by activating endogenous regeneration ^[40][121] or through cell transplantation ^[39][120,122]. One approach to treating a number of degenerative retinal diseases, including age-related macular degeneration, is cell replacement therapy. RPE cells derived from human embryonic SCs and iPSCs are already undergoing clinical trials and have great promise for the treatment of both age-related macular degeneration ^[41][123,124,125] and hereditary RPE-associated retinal dystrophies ^[42][126]. Despite the highly effective protocols for obtaining RPE cells in sufficient quantities for transplantations that have been developed, they still remain labor- and time-consuming ^[43][127]. In addition, there is the problem of immunosuppression and tumorigenesis ^[44][128,129], and moral and ethical issues regarding the use of ESCs remain unresolved ^[45][130]. Unlike

В отличие от ESCs andи iPSCs, adult human RPESCs, despite their limited proliferative potential, do not form взрослого человека, несмотря на их ограниченный пролиферативный потенциал, не образуют опухоли [ tumors100 ^[12]]. RPESCsРПЭСК are capable of producing RPE-RPESC progeny, which corresponds to the native RPE in its morphologicalспособны производить потомство РПЭ-РПЭС, которое по морфологическим и функциональным характеристикам соответствует нативному РПЭ [ and95 functional, characteristics97 ^[15]., The99 preclinical, transplantation113 of, RPE114 derived]. from RPESCs has shown that an intermediate stage of RPE differentiation is more effective in restoring visionДоклиническая трансплантация РПЭ, полученного из РПЭСК, показала, что промежуточная стадия дифференцировки РПЭ более эффективна для восстановления зрения ^[16].[ The97 successful]. transplantation of humanУспешная трансплантация человеческих fRPESCs has been demonstrated on animal models, where the cells differentiate into both RPE and photoreceptorsбыла продемонстрирована на животных моделях, где клетки дифференцировались как в RPE, так и ^[17]. Theв heterogeneityфоторецепторы of[ RPE-RPESCs27 is].

Гетерогенность manifestedРПЭ-РПЭСК not only in the functional specialization of cells, but also in the ability of RPE-RPESCs to successfully transplant. Several subpopulations ofпроявляется не только в функциональной специализации клеток, но и в способности РПЭ-РПЭСК успешно трансплантироваться. Было идентифицировано несколько субпопуляций RPE-RPESCs have been identified that may be potential candidates for, которые могут быть потенциальными кандидатами на эффективную трансплантацию [ effective71 transplantation ^[30]]. TheseЭти cells showed the enrichment of signaling pathways associated with cell differentiation and proliferation. Nevertheless, among them, only one cluster ofклетки показали обогащение сигнальных путей, связанных с дифференцировкой и пролиферацией клеток. Тем не менее, среди них был выделен только один кластер EZH2-positive RPE-RPESCs was isolated, which is capable of integrating into the host RPE monolayer upon transplantation. A clear difference between the transcriptomes of transplant-effective and transplant-ineffective subpopulations of позитивных РПЭ-РПЭСК, способный интегрироваться в монослой РПЭ хозяина при трансплантации. Выявлена четкая разница между транскриптомами трансплантоэффективных и трансплантонеэффективных субпопуляций RPE-RPESCs was identified. In addition, molecular pathways associated with graft effectiveness were shown, and potential biomarkers for effective RPE cultures were. Кроме того, были показаны молекулярные пути, связанные с эффективностью трансплантата, и установлены потенциальные биомаркеры для эффективных культур РПЭ [ established71 ^[30]]. AsВ a primary biomarker of transplantation success, the long noncoding RNA (lncRNAкачестве первичного биомаркера успеха трансплантации рассматривалась длинная некодирующая РНК (днРНК) Three Prime Repair Exonuclease 1 (TREX) was considered, which regulates various cellular processes, including migration and cell survival, которая регулирует различные клеточные процессы, включая миграцию и выживание клеток ^[30].[ These71 data]. will allow us to identify from the heterogeneous populations of RPESC-RPE, iPSC-RPE, ESC-RPE, and RPE obtained from other possible cell sources a separate subpopulation of cells that is mostЭти данные позволят нам выделить из гетерогенных популяций РПЭСК-РПЭ, иПСК-РПЭ, ЭСК-РПЭ и РПЭ, полученных из других возможных источников клеток, отдельную субпопуляцию клеток, наиболее подходящую для успешной трансплантации.

В suitableнастоящее for successful transplantation. Currently, время RPE, derived fromполученный из RPESCs represents a potentially unlimited source of HLA-compatible cells and an unlimited donor source with several qualities favorable for transplantation, including stability, ubiquity, and cost. In addition, active phase 1 clinical trials are being carried out in patients with dry AMD , представляет собой потенциально неограниченный источник HLA-совместимых клеток и неограниченный источник доноров с несколькими качествами, благоприятными для трансплантации, включая стабильность, повсеместное распространение и стоимость. Кроме того, активные клинические исследования фазы 1 проводятся у пациентов с сухой ВМД (NCT04627428) ^[46].[ It131 is]. important to note that transplantation can cause a number of complications, which can be triggered by surgical damage to the retina, leading toВажно отметить, что трансплантация может вызвать ряд осложнений, которые могут быть спровоцированы хирургическим повреждением сетчатки, приводящим к ее отслойке [ its132 detachment ^[47]]. InВ this regard, the stimulation of endogenous adult RPESCs present in the eye to produce a new autologous RPE связи с этим стимуляция эндогенных взрослых РПЭСК, присутствующих в глазу, для получения нового аутологичного РПЭ in situ without surgery may be promising for the treatment of many degenerative-dystrophic diseases of the human retina and will help to avoid the above limitations and complicationsбез хирургического вмешательства может быть перспективной для лечения многих дегенеративно-дистрофических заболеваний сетчатки человека и поможет избежать вышеперечисленных ограничений и осложнений. .