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Sarveiskalvo on silmän etuosaa suojaava läpinäkyvä kudos, jolla on keskeinen rooli näkökyvyn mahdollistamisessa. Sen uloin osa koostuu kerrostuneesta, jatkuvassa uusiutumissyklissä olevasta epiteelisolukosta. Tätä jatkuvaa uusiutumista ylläpitävät limbaalisiksi kantasoluiksi (engl. limbal stem cells, LSC) kutsutut aikuiset, kudosspesifiset kantasolut, joiden jakautuminen tuottaa uusia sarveiskalvoepiteelin soluja läpi ihmisen elämän. LSC-solut sijaitsevat hyvin suojattuina sarveiskalvon ja sidekalvon rajapinnassa, jonka erityinen anatominen mikroympäristö huolehtii tarkasti niiden ominaisuuksien säilymisestä ja toiminnan säätelemisestä. Limbaalisten kantasolujen puutos (engl. limbal stem cell deficiency, LSCD) on toimintakykyä ja hyvinvointia rajusti heikentävä, sokeuttava silmäsairaus, joka johtuu häiriöstä LSC-solujen toiminnassa ja sen myötä sarveiskalvon pinnan uusiutumisessa. Hoitona toimii ainoastaan uusien, toimivien LSC-solujen vieminen sarveiskalvolle. Sopivista LSC-solusiirteistä, joita voidaan saada joko potilaan omasta terveestä silmästä tai ulkopuoliselta luovuttajalta, on kuitenkin jatkuvasti pulaa, mikä on kasvattanut kiinnostusta vaihtoehtoisia solulähteitä kohtaan. Ihmisen erittäin monikykyisillä kantasoluilla (engl. human pluripotent stem cells, hPSC) on kyky paitsi rajattomaan uusiutumiseen, myös kyky erilaistumiseen sarveiskalvoepiteelin soluiksi. Nämä ominaisuudet tekevät hPSC-soluista erittäin viehättävän työkalun uusien solukorvaushoitojen kehittämisessä. Haasteita kentälle luovat LSC-solujen tehokkaiden tunnistamis- ja rikastamismenetelmien puute, sillä LSC-siirteiden hoitoteho on pääosin riippuvainen siirteen sisältämien aitojen kantasolujen määrästä. LSCD:stä kärsivien potilaiden hoidon parantamiseksi kaivataan menetelmiä, joilla sarveiskalvon uusiutumiskyvyn palauttavia LSC-soluja pystyttäisiin tuottamaan tehokkaasti paitsi kliinisesti yhteensopivilla myös taloudellisesti toteuttamiskelpoisilla menetelmillä. Tässä väitöskirjatutkimuksessa paneuduttiin yllä mainittuun tarpeeseen ja tutkittiin kustannustehokkaita, eläinperäisiin komponentteihin nojaamattomia menetelmiä toiminnallisten LSC-soluja tuottamiseksi hPSC-soluista. Tutkimusprojektin ensimmäisissä osissa keskityttiin optimoimaan tukisoluttomia, kliiniseen käyttöön sovitettavissa olevia hPSC-solujen kasvatus- ja erilaistusmenetelmiä sekä karakterisoimaan sarveiskalvoerilaistuksen seurauksena saatavien LSC-soluja muistuttavien solujen ilmiasua. Tutkimuksen seuraavissa vaiheissa hPSC-LSC-solujen erilaistumisprosessia tarkasteltiin entistä yksityiskohtaisemmin ja tunnistettiin myös varhaisemman ilmiasun omaava, ohimenevästi ilmenevä alapopulaatio. Tämän erittäin korkean uusiutumiskyvyn omaavan ja sarveiskalvon kudosmallissa lupaavasti käyttäytyvän populaation ilmiasu saatiin säilytettyä väitöskirjatyön aikana kehitetyillä uusilla, erilaistumista hillitsevillä soluviljelymenetelmillä. Viimeisessä työssä myös verrattiin hPSC-LSC-soluja kudoksesta eristettyihin vastaaviin soluihin sekä arvioitiin alustavasti niiden immunologisia ominaisuuksia ja kudoksen korjauskykyä soluviljelyolosuhteissa. Kaiken kaikkiaan tämä väitöskirja esittelee tehokkaita, sekä taloudellisesti että kliinisesti toteuttamiskelpoisia ratkaisuja toiminnallisten LSC-solujen erilaistamiseksi rajattoman lähtömateriaalin tarjoavista hPSC-soluista. Tässä väitöskirjassa esitellyt tulokset edistävät uusien, kehittyneiden kantasoluperäisten hoitomuotojen saatavuutta sarveiskalvon pinnan vakavista vaurioista kuten LSCD:stä kärsiville potilaille. Lisäksi ne ovat lisänneet tietoa hPSC-LSC-solujen erilaistumisprosessista ja terapeuttisesti kiinnostavien alapopulaatioiden ominaisuuksista, luoden hyvän pohjan menetelmien jatkokehittämiselle. Cornea is the transparent tissue covering the front of the eye and has an integral role in enabling vision. The outermost structure of the cornea is the multilayered epithelium, which maintains the corneal homeostasis via constant turnover of the epithelial cells. This dynamic renewal process is enabled by adult tissue-specific stem cells termed the limbal stem cells (LSCs), which supply the cornea with new cells throughout life. The LSCs are protected from outside hazards by their anatomically specialized niche environment, which also provides the necessary cues to regulate their function. Limbal stem cell deficiency (LSCD) is a debilitating ocular surface disorder resulting from the loss of or severe disturbance in LSC function. Its ultimate manifestation is blindness. The only curative treatment option involves re- establishment of the corneal renewal capacity via introduction of new, functional LSCs. However, shortage of suitable autologous or allogeneic LSCs has prompted the research on alternative cell sources, including human pluripotent stem cells (hPSCs). Human PSCs have an unlimited self-renewal capacity and can be differentiated towards corneal cells, which renders them an attractive tool for the use of ocular surface regenerative therapies. The success of the LSC grafts is dependent on the presence of true stem cells, whose definite identification and subsequent enrichment have remained a challenge. Economically feasible, robust protocols for yielding LSCs under clinically applicable conditions are demanded to promote the treatment of patients suffering from the LSCD. The work presented in this dissertation sought to develop methods for the differentiation of therapeutic LSCs from hPSCs using a clinically upgradable, feeder- independent, and chemically defined setting. The first parts of the thesis work concentrated on establishing feeder-independent conditions for the undifferentiated hPSCs, followed by the optimization of the corneal differentiation and cryopreservation protocols, and phenotypic characterization of the derivative cells. During the project, the characterization proceeded to the detailed dissection of the hPSC-LSC differentiation process and identification of subpopulations displaying distinct LSC-associated marker protein profiles. Novel culture conditions were successfully established to halt the differentiation of hPSC-LSCs to a specific state, which was shown to promote the hPSC-LSC self-renewal and ex vivo regenerative capabilities. Finally, the hPSC-LSCs were compared with their tissue-derived counterparts and preliminarily evaluated for their immunogenicity and wound healing properties in vitro. To conclude, this dissertation describes efficient, feasible, and, most importantly, clinically applicable techniques for the differentiation of hPSCs towards therapeutically potent LSCs. The results presented in this dissertation have contributed to developing achievable hPSC-based methods for advanced corneal cell therapy applications for patients suffering from LSDC. Moreover, they have increased the knowledge regarding the hPSC-LSC differentiation process and various therapeutically interesting subpopulations of hPSC-LSCs, laying a firm foundation for further investigations.

Additional file 2: Containing supplementary data about CK3 and CK12 expression in post-thaw p1 hPSC-LSCs (Supplementary Fig. S1), ���Np63�� expression in hPSC-LSCs derived from the ABCG2+ cell population (Supplementary Fig. S2) and the Supplementary Movie S3 showing the wound closure of p1 hPSC-derived LSCs versus the tissue-derived porcine LSCs

Figure S1. Description of three distinct stem cell population phenotypes emerging during the hPSC-LSC differentiation, separated by their expression patterns for ABCG2 and ∆Np63α. Representative cells from different populations are marked with symbols to the IF images of d10 and d24 cytospin samples. Scale bars, 100 μm. Cell nuclei counterstained with DAPI (blue). Data are presented with the representative hESC line Regea08/017. (DOCX 742 kb)

Figure S2. (A) Representative flow cytometry graphs of the negative controls, isotype controls, and ABCG2-stained hPSC-LSC samples in different time points. (B) Morphology and ABCG2/p63α expression of day 11 sorted ABCG2-positive hPSC-LSCs after continued culture (17 days) in CnT-30 medium and on LN-521/Col IV (B). Scale bar, 100 μm. Cell nuclei counterstained with DAPI (blue). BF: brightfield, FACS: fluorescence-activated cell sorting. Data are presented with the representative hESC line Regea08/017. (DOCX 1668 kb)

Figure S4. (A) Additional characterization of OCT3/4, PAX6, CK14, and CK15 expression at d24 in the novel CnT-07+ENRC maintenance condition. (B) Characterization of Ki67/ABCG2 protein expression at d11, as well as after continued culture in CnT-07+ENRC or CnT-30 at d21. In both panels, cell nuclei counterstained with DAPI (blue) and scale bars, 100â Îźm. Data are presented for the hESC lines Regea11/013 (A) and Regea08/017 (B). (DOCX 5938 kb)

Supplementary materials and methods. (DOCX 65 kb)

(DOCX 19 kb)

Supplementary dataset 1. Optimization of hPSC-RPE differentiation. (DOCX 36 kb)

Supplementary dataset 2. Optimization of hPSC-LESC differentiation. (DOCX 42 kb)

Figure S3. Characterization of putative LSC marker expression during hPSC-LSC differentiation for hiPSC line UTA.04607.WT. (A) Representative morphology and protein expression of the cultures at selected time points. Scale bars, 100 μm for all images in the same column. Cell nuclei counterstained with DAPI (blue). (B) Marker expression differences in the d10 and d24 populations. Five images per sample and a minimum of 600 cells per time point were analyzed for each marker from cytospin samples. (C) p63α and ABCG2 expression in d10 and d24 hPSC-LSCs. Five images per sample and a minimum of 3 000 cells per time point were analyzed from cytospin samples. (D) The level of ABCG2 protein expression in UD-hPSCs and in d10 and d24–26 hPSC-LSCs, analyzed with flow cytometry. (G) The ABCG2 mRNA expression levels in UD-hPSCs and in d10 and d24 hPSC-LSCs analyzed with qRT-PCR. All quantitative data are presented as the mean + SD and n marks the individual cell differentiation batches serving as biological replicates. Statistical analysis in (D) was carried out using the Mann-Whitney U test. *P ≤ 0.05. (DOCX 4413 kb)

Table S2. Distribution of ABCG2-positive and ABCG2-negative cells in Regea08/017 d10 hPSC-LSC subpopulations marked by selected cell surface markers. (DOCX 19 kb)

Figure S3. Characterization of putative LSC marker expression during hPSC-LSC differentiation for hiPSC line UTA.04607.WT. (A) Representative morphology and protein expression of the cultures at selected time points. Scale bars, 100 μm for all images in the same column. Cell nuclei counterstained with DAPI (blue). (B) Marker expression differences in the d10 and d24 populations. Five images per sample and a minimum of 600 cells per time point were analyzed for each marker from cytospin samples. (C) p63α and ABCG2 expression in d10 and d24 hPSC-LSCs. Five images per sample and a minimum of 3 000 cells per time point were analyzed from cytospin samples. (D) The level of ABCG2 protein expression in UD-hPSCs and in d10 and d24–26 hPSC-LSCs, analyzed with flow cytometry. (G) The ABCG2 mRNA expression levels in UD-hPSCs and in d10 and d24 hPSC-LSCs analyzed with qRT-PCR. All quantitative data are presented as the mean + SD and n marks the individual cell differentiation batches serving as biological replicates. Statistical analysis in (D) was carried out using the Mann-Whitney U test. *P ≤ 0.05. (DOCX 4413 kb)

Full detailed descriptions for the hPSC differentiation towards LSCs and standard hPSC-LSC culture, establishment and maintenance of the ABCG2-positive hPSC-LSC culture, immunofluorescence characterization protocol, flow cytometry analysis and fluorescence-activated cell sorting protocol, quantitative RT-PCR protocol, and cell surface antigen screening with the LEGENDScreenâ ˘ Kit. (DOCX 29 kb)

Table S1. Primary and secondary antibodies used in this study. (DOCX 22 kb)

Table S2. Distribution of ABCG2-positive and ABCG2-negative cells in Regea08/017 d10 hPSC-LSC subpopulations marked by selected cell surface markers. (DOCX 19 kb)

Full detailed descriptions for the hPSC differentiation towards LSCs and standard hPSC-LSC culture, establishment and maintenance of the ABCG2-positive hPSC-LSC culture, immunofluorescence characterization protocol, flow cytometry analysis and fluorescence-activated cell sorting protocol, quantitative RT-PCR protocol, and cell surface antigen screening with the LEGENDScreenâ ˘ Kit. (DOCX 29 kb)

Table S1. Primary and secondary antibodies used in this study. (DOCX 22 kb)

Additional file 1: Containing detailed descriptions of the fluorescence activated cell sorting of ABCG2+ hPSC-LSCs and further differentiation of p1 hPSC-LSCs

Additional file 1: Containing detailed descriptions of the fluorescence activated cell sorting of ABCG2+ hPSC-LSCs and further differentiation of p1 hPSC-LSCs