{"id":{"repo_id":"helsinki","oai_identifier":"oai:helda.helsinki.fi:10138/591573"},"canonical_url":"https://search.dev.ndltd.org/etd/helsinki/oai:helda.helsinki.fi:10138/591573","repository":{"repo_id":"helsinki","name":"University of Helsinki","base_url":"https://helda.helsinki.fi/server/oai/request"},"display":{"title":"Stem Cell-Based Immune Modulation and Regeneration","abstract":"Tendinopathies, including both degenerative and inflammatory-based diseases of tendon tissue, include a significant clinical challenge, impacting millions worldwide and imposing a substantial socio-economic burden. Tendinopathies are often chronic conditions induced by an unsolved healing process hesitating in the persistence of active immune cells such as macrophages, neutrophils, and lymphocytes, which release pro-inflammatory cytokines and matrix-degrading enzymes. The sustained inflammatory phase, aggravated by tendons’ inherently low regenerative properties, hinders tissue repair and leads to a poorly organized extracellular matrix, impairing tissue biomechanical properties. Current treatments primarily offer symptom relief without addressing the root inflammatory mechanisms driving the pathology and occurrence of recurrence. This thesis explores the mechanisms behind the emerging pro-tenogenic properties of amniotic epithelial cells (AEC), primarily mediated by their paracrine release of immunomodulatory molecules. The underlying hypothesis proposes that stem cells’ bioactive molecules promote tendon regeneration by facilitating an early shift from the inflammatory phase to the proliferative healing phase. Based on this premise, the thesis aimed to produce AEC immune regulatory secretomes to use them as innovative stem cell-free solutions for controlling the early immune phase of tissue regeneration. The first step assessed whether the immunomodulatory properties of AEC depend on cell phenotype, considering their ability to undergo Epithelial-Mesenchymal Transition (EMT) to support regeneration or interact with different materials. In this context, we found that AEC promptly activated EMT when seeded on graphene oxide (GO)-coated surfaces, a carbon-based nanomaterial that has entered the field with several biomedical applications owing to its exceptional physicochemical and biological features. The cells interacted with GO by activating a specific TGF-β/SMAD signaling pathway that enhances some pro-regenerative AEC properties such as cell migration and adhesion but simultaneously diminishes their ability to inhibit immune cell activation (PBMC), thus confirming that when AEC shift toward the mesenchymal phenotype, their ability to modulate immune responses through paracrine signaling decreases. Subsequently, the study moved towards investigating the molecular mechanism that underlies the paracrine immunomodulatory properties that AEC can express both intrinsically and following exposure to external stimuli. AEC-derived amphiregulin (AREG)/EGFR axis was identified as a key intracellular pathway in regulating immune responses. The activation of this powerful immune-modulatory factor is controlled through the COX-2/PGE2/EP4 axis, which can be activated from different and sometimes converging pathways. First, LPS promotes it through NF-κβ activation but induces YAP degradation, while stretching promotes it through YAP activity. Combined, LPS suppresses stretching's effects, emphasizing the need to balance these stimuli for fine-tuning their anti-inflammatory effects. Beyond cellular responses, this thesis delved into the fractions of the AEC immune modulatory secretome. To this aim, microvesicles (MV) and MV-free fractions were isolated, characterized, and studied for their modulating immune function. It was found that AEC exert their immune modulatory activities using both MV-free and MV secretome fractions. For the first time, we demonstrated that the MV fraction of AEC secretome is enriched of organelle cargo (mitochondria, RE) that can be actively internalized in immune cells, triggering mitochondria-dependent apoptosis and thereby highlighting a potent, cell-free immune suppressive mechanism that could offer therapeutic advantages without the need for direct cell transplantation. Interestingly, the MV-free fraction of the AEC secretome was more effective in inhibiting PBMC and Jurkat cell activation in response to LPS stimuli by rapidly increasing its soluble immunomodulatory components. To translate these findings into potential clinical treatments, the AEC secretome was then encapsulated within a Hyaluronic Acid- Polyethylene Glycol-Heparin (HA-PEG-HEP) hydrogel matrix, which provided a controlled and sustained release of bioactive molecules, significantly bolstering both immunosuppressive and pro-regenerative effects over extended periods. This encapsulation strategy preserved the bioactivity of crucial components like AREG, advancing the therapeutic potential of AEC secretome in a practical, scalable form suitable for clinical use. By harnessing AEC's ability to modulate immune responses and support tissue repair, this dissertation lays the groundwork for the development of cell-free therapies that have broad implications for managing inflammation and degenerative conditions, marking a significant step toward translating AEC-based regenerative therapies from bench to bedside.","abstract_html":"Tendinopathies, including both degenerative and inflammatory-based diseases of tendon tissue, include a significant clinical challenge, impacting millions worldwide and imposing a substantial socio-economic burden. Tendinopathies are often chronic conditions induced by an unsolved healing process hesitating in the persistence of active immune cells such as macrophages, neutrophils, and lymphocytes, which release pro-inflammatory cytokines and matrix-degrading enzymes. The sustained inflammatory phase, aggravated by tendons’ inherently low regenerative properties, hinders tissue repair and leads to a poorly organized extracellular matrix, impairing tissue biomechanical properties. Current treatments primarily offer symptom relief without addressing the root inflammatory mechanisms driving the pathology and occurrence of recurrence. This thesis explores the mechanisms behind the emerging pro-tenogenic properties of amniotic epithelial cells (AEC), primarily mediated by their paracrine release of immunomodulatory molecules. The underlying hypothesis proposes that stem cells’ bioactive molecules promote tendon regeneration by facilitating an early shift from the inflammatory phase to the proliferative healing phase. Based on this premise, the thesis aimed to produce AEC immune regulatory secretomes to use them as innovative stem cell-free solutions for controlling the early immune phase of tissue regeneration. The first step assessed whether the immunomodulatory properties of AEC depend on cell phenotype, considering their ability to undergo Epithelial-Mesenchymal Transition (EMT) to support regeneration or interact with different materials. In this context, we found that AEC promptly activated EMT when seeded on graphene oxide (GO)-coated surfaces, a carbon-based nanomaterial that has entered the field with several biomedical applications owing to its exceptional physicochemical and biological features. The cells interacted with GO by activating a specific TGF-β/SMAD signaling pathway that enhances some pro-regenerative AEC properties such as cell migration and adhesion but simultaneously diminishes their ability to inhibit immune cell activation (PBMC), thus confirming that when AEC shift toward the mesenchymal phenotype, their ability to modulate immune responses through paracrine signaling decreases. Subsequently, the study moved towards investigating the molecular mechanism that underlies the paracrine immunomodulatory properties that AEC can express both intrinsically and following exposure to external stimuli. AEC-derived amphiregulin (AREG)/EGFR axis was identified as a key intracellular pathway in regulating immune responses. The activation of this powerful immune-modulatory factor is controlled through the COX-2/PGE2/EP4 axis, which can be activated from different and sometimes converging pathways. First, LPS promotes it through NF-κβ activation but induces YAP degradation, while stretching promotes it through YAP activity. Combined, LPS suppresses stretching's effects, emphasizing the need to balance these stimuli for fine-tuning their anti-inflammatory effects. Beyond cellular responses, this thesis delved into the fractions of the AEC immune modulatory secretome. To this aim, microvesicles (MV) and MV-free fractions were isolated, characterized, and studied for their modulating immune function. It was found that AEC exert their immune modulatory activities using both MV-free and MV secretome fractions. For the first time, we demonstrated that the MV fraction of AEC secretome is enriched of organelle cargo (mitochondria, RE) that can be actively internalized in immune cells, triggering mitochondria-dependent apoptosis and thereby highlighting a potent, cell-free immune suppressive mechanism that could offer therapeutic advantages without the need for direct cell transplantation. Interestingly, the MV-free fraction of the AEC secretome was more effective in inhibiting PBMC and Jurkat cell activation in response to LPS stimuli by rapidly increasing its soluble immunomodulatory components. To translate these findings into potential clinical treatments, the AEC secretome was then encapsulated within a Hyaluronic Acid- Polyethylene Glycol-Heparin (HA-PEG-HEP) hydrogel matrix, which provided a controlled and sustained release of bioactive molecules, significantly bolstering both immunosuppressive and pro-regenerative effects over extended periods. This encapsulation strategy preserved the bioactivity of crucial components like AREG, advancing the therapeutic potential of AEC secretome in a practical, scalable form suitable for clinical use. By harnessing AEC's ability to modulate immune responses and support tissue repair, this dissertation lays the groundwork for the development of cell-free therapies that have broad implications for managing inflammation and degenerative conditions, marking a significant step toward translating AEC-based regenerative therapies from bench to bedside.","abstract_has_math":false,"creators":["Cerveró Varona, Adrián"],"institution":"University of Helsinki","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-02-25","date_published":"2025-02-25","updated_at":"2026-08-21T22:21:56Z","subjects":["drug Research"],"languages":["eng"],"rights":["This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.","Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.","Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10138/591573","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"source_record":{"url":"https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F591573","prefix":"dim"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Cerveró Varona, Adrián"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-01-30T12:18:30Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-01-30T12:18:30Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-02-25"]},{"key":"dc:publisher","label":"Institution","values":["University of Helsinki","Helsingin yliopisto","Helsingfors universitet"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["drug Research"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.","Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.","Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10138/591573"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Tendinopathies, including both degenerative and inflammatory-based diseases of tendon tissue, include a significant clinical challenge, impacting millions worldwide and imposing a substantial socio-economic burden. Tendinopathies are often chronic conditions induced by an unsolved healing process hesitating in the persistence of active immune cells such as macrophages, neutrophils, and lymphocytes, which release pro-inflammatory cytokines and matrix-degrading enzymes. The sustained inflammatory phase, aggravated by tendons’ inherently low regenerative properties, hinders tissue repair and leads to a poorly organized extracellular matrix, impairing tissue biomechanical properties. Current treatments primarily offer symptom relief without addressing the root inflammatory mechanisms driving the pathology and occurrence of recurrence. This thesis explores the mechanisms behind the emerging pro-tenogenic properties of amniotic epithelial cells (AEC), primarily mediated by their paracrine release of immunomodulatory molecules. The underlying hypothesis proposes that stem cells’ bioactive molecules promote tendon regeneration by facilitating an early shift from the inflammatory phase to the proliferative healing phase. Based on this premise, the thesis aimed to produce AEC immune regulatory secretomes to use them as innovative stem cell-free solutions for controlling the early immune phase of tissue regeneration. The first step assessed whether the immunomodulatory properties of AEC depend on cell phenotype, considering their ability to undergo Epithelial-Mesenchymal Transition (EMT) to support regeneration or interact with different materials. In this context, we found that AEC promptly activated EMT when seeded on graphene oxide (GO)-coated surfaces, a carbon-based nanomaterial that has entered the field with several biomedical applications owing to its exceptional physicochemical and biological features. The cells interacted with GO by activating a specific TGF-β/SMAD signaling pathway that enhances some pro-regenerative AEC properties such as cell migration and adhesion but simultaneously diminishes their ability to inhibit immune cell activation (PBMC), thus confirming that when AEC shift toward the mesenchymal phenotype, their ability to modulate immune responses through paracrine signaling decreases. Subsequently, the study moved towards investigating the molecular mechanism that underlies the paracrine immunomodulatory properties that AEC can express both intrinsically and following exposure to external stimuli. AEC-derived amphiregulin (AREG)/EGFR axis was identified as a key intracellular pathway in regulating immune responses. The activation of this powerful immune-modulatory factor is controlled through the COX-2/PGE2/EP4 axis, which can be activated from different and sometimes converging pathways. First, LPS promotes it through NF-κβ activation but induces YAP degradation, while stretching promotes it through YAP activity. Combined, LPS suppresses stretching's effects, emphasizing the need to balance these stimuli for fine-tuning their anti-inflammatory effects. Beyond cellular responses, this thesis delved into the fractions of the AEC immune modulatory secretome. To this aim, microvesicles (MV) and MV-free fractions were isolated, characterized, and studied for their modulating immune function. It was found that AEC exert their immune modulatory activities using both MV-free and MV secretome fractions. For the first time, we demonstrated that the MV fraction of AEC secretome is enriched of organelle cargo (mitochondria, RE) that can be actively internalized in immune cells, triggering mitochondria-dependent apoptosis and thereby highlighting a potent, cell-free immune suppressive mechanism that could offer therapeutic advantages without the need for direct cell transplantation. Interestingly, the MV-free fraction of the AEC secretome was more effective in inhibiting PBMC and Jurkat cell activation in response to LPS stimuli by rapidly increasing its soluble immunomodulatory components. To translate these findings into potential clinical treatments, the AEC secretome was then encapsulated within a Hyaluronic Acid- Polyethylene Glycol-Heparin (HA-PEG-HEP) hydrogel matrix, which provided a controlled and sustained release of bioactive molecules, significantly bolstering both immunosuppressive and pro-regenerative effects over extended periods. This encapsulation strategy preserved the bioactivity of crucial components like AREG, advancing the therapeutic potential of AEC secretome in a practical, scalable form suitable for clinical use. By harnessing AEC's ability to modulate immune responses and support tissue repair, this dissertation lays the groundwork for the development of cell-free therapies that have broad implications for managing inflammation and degenerative conditions, marking a significant step toward translating AEC-based regenerative therapies from bench to bedside.","Jännetautien, joihin kuuluvat sekä rappeuttavat että tulehdukselliset jännekudoksen sairaudet, muodostavat merkittävän kliinisen haasteen, vaikuttaen miljooniin ihmisiin maailmanlaajuisesti ja aiheuttaen huomattavan sosioekonomisen taakan. Jännetautien kehittyminen on usein krooninen prosessi, jossa parantumisprosessi jää kesken, mikä johtaa immuunisolujen, kuten makrofagien, neutrofiilien ja lymfosyyttien, jatkuvaan aktivoitumiseen. Nämä solut vapauttavat tulehdusta edistäviä sytokiineja ja matriksia hajottavia entsyymejä. Pitkittynyt tulehdusvaihe, yhdistettynä jännekudoksen alhaiseen uusiutumiskykyyn, estää kudoksen korjaantumista ja johtaa epäjärjestäytyneeseen soluväliaineeseen, heikentäen kudoksen biomekaanisia ominaisuuksia. Nykyiset hoitokeinot lievittävät lähinnä oireita, mutta eivät kohdenna sairauden taustalla olevia tulehduksellisia mekanismeja, jotka aiheuttavat patologian ja lisäävät uusiutumisriskiä. Tämä väitöskirja tutkii amnionin epiteelisolujen (AEC) uusia pro-tenogeenisiä ominaisuuksia, jotka välittyvät ensisijaisesti niiden parakriinisten immunomodulatoristen molekyylien kautta. Perusolettamuksena on, että kantasolujen bioaktiiviset molekyylit edistävät jänteen uudistumista helpottamalla tulehdusvaiheen varhaista siirtymistä lisääntymisvaiheeseen. Tämän pohjalta väitöskirjan tavoitteena oli tuottaa AEC:n immunomodulatorisia eritystuotteita ja hyödyntää niitä uusina kantasoluttomina ratkaisuina kudoksen uudistumisen varhaisen immuunivaiheen hallintaan. Ensimmäinen vaihe oli arvioida, riippuuko AEC:n immunomodulatorinen potentiaali solujen fenotyypistä, kun otetaan huomioon niiden kyky käydä läpi epiteeli-mesenkymaali transitiota (EMT) kudoksen uusiutumisen tukemiseksi tai niiden vuorovaikutus eri materiaalien kanssa. Tässä yhteydessä havaitsimme, että AEC käynnistivät nopeasti EMT:n, kun ne kylvettiin grafeenioksidilla (GO) pinnoitetuille pinnoille – hiilipohjainen nanomateriaali, jolla on useita biolääketieteellisiä sovelluksia erinomaisen fysiokemiallisen ja biologisen ominaisuutensa ansiosta. Solut vuorovaikuttivat GO:n kanssa aktivoimalla TGF-β/SMAD-signaalireitin, joka parantaa tiettyjä AEC:n pro-regeneratiivisia ominaisuuksia, kuten solujen migraatiota ja adheesiota, mutta samanaikaisesti vähentää niiden kykyä estää immuunisolujen aktivoitumista (PBMC). Tämä vahvisti, että kun AEC siirtyvät mesenkymaaliseksi fenotyypiksi, niiden kyky säädellä immuunivastetta parakriinisen signaloinnin kautta heikkenee. Seuraavaksi tutkimuksessa selvitettiin molekyylimekanismeja, jotka säätelevät AEC:n parakriinista immunomodulatorista vaikutusta sekä luontaisesti että ulkoisten ärsykkeiden seurauksena. AEC:n erittämän amfireguliinin (AREG)/EGFR-akselin havaittiin olevan keskeinen immuunivasteiden säätelyssä. Tämän voimakkaan immunomodulatorisen tekijän aktivointi tapahtuu COX-2/PGE2/EP4-reitin kautta, joka voi aktivoitua useista eri ja joskus yhdistyvistä reiteistä. Esimerkiksi LPS edistää sen aktivaatiota NF-κB:n kautta mutta samalla hajottaa YAP:ia, kun taas venytys edistää YAP-aktiivisuutta. Yhdessä LPS estää venytyksen vaikutuksia, mikä korostaa tarvetta tasapainottaa näitä ärsykkeitä niiden tulehdusta estävien vaikutusten hienosäätämiseksi. Soluvastausten lisäksi tutkimuksessa keskityttiin AEC:n immunomodulatorisen eritystuotteen osajakeisiin. Tätä varten mikrovesikkelit (MV) ja MV-vapaat jakeet eristettiin, karakterisoitiin ja tutkittiin niiden immuunivasteita säätelevän vaikutuksen osalta. Havaittiin, että AEC toteuttavat immunomodulatoriset vaikutuksensa sekä MV-vapaan että MV-eritystuotejakeiden kautta. Ensimmäistä kertaa osoitettiin, että AEC:n eritystuotteiden MV-jakeet sisältävät organelleja (mitokondrioita, RE), jotka voivat aktiivisesti siirtyä immuunisoluihin, laukaisten mitokondrioriippuvaisen apoptoosin. Tämä paljasti voimakkaan, soluttoman immuunisuppressiivisen mekanismin, joka voi tarjota terapeuttisia etuja ilman suoraa solusiirtoa. Kiinnostavasti havaittiin, että MV-vapaa eritystuotejakso oli tehokkaampi PBMC:n ja Jurkat-solujen aktivaation estämisessä LPS-ärsykkeeseen vastaamalla, sillä se lisäsi nopeasti liukoisten immunomodulatoristen komponenttien määrää. Näiden löydösten kliinistä soveltamista varten AEC:n eritystuote kapseloitiin hyaluronihappo-polyetyleeniglykoli-hepariini (HA-PEG-HEP) -hydrogeelimatriksiin, joka tarjosi kontrolloidun ja pitkäkestoisen bioaktiivisten molekyylien vapautumisen, vahvistaen merkittävästi sekä immunosuppressiivisia että pro-regeneratiivisia vaikutuksia pitkällä aikavälillä. Tämä kapselointistrategia säilytti kriittisten komponenttien, kuten AREG:n, bioaktiivisuuden, edistäen AEC-eritystuotteiden terapeuttista potentiaalia kliinisessä, skaalautuvassa muodossa. Hyödyntämällä AEC:n kykyä säädellä immuunivasteita ja tukea kudoksen uusiutumista, tämä väitöskirja luo perustan soluttomille terapioille, joilla on laaja-alaisia vaikutuksia tulehduksellisten ja rappeuttavien sairauksien hallintaan. Tämä merkitsee merkittävää askelta kohti AEC-pohjaisten regeneratiivisten hoitojen viemistä laboratoriosta kliiniseen käyttöön."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Stem Cell-Based Immune Modulation and Regeneration"]}]}],"canonical_facts":{"dc:creator":["Cerveró Varona, Adrián"],"dc:date.accessioned":["2025-01-30T12:18:30Z"],"dc:date.available":["2025-01-30T12:18:30Z"],"dc:date.issued":["2025-02-25"],"dc:description.abstract":["Tendinopathies, including both degenerative and inflammatory-based diseases of tendon tissue, include a significant clinical challenge, impacting millions worldwide and imposing a substantial socio-economic burden. Tendinopathies are often chronic conditions induced by an unsolved healing process hesitating in the persistence of active immune cells such as macrophages, neutrophils, and lymphocytes, which release pro-inflammatory cytokines and matrix-degrading enzymes. The sustained inflammatory phase, aggravated by tendons’ inherently low regenerative properties, hinders tissue repair and leads to a poorly organized extracellular matrix, impairing tissue biomechanical properties. Current treatments primarily offer symptom relief without addressing the root inflammatory mechanisms driving the pathology and occurrence of recurrence. This thesis explores the mechanisms behind the emerging pro-tenogenic properties of amniotic epithelial cells (AEC), primarily mediated by their paracrine release of immunomodulatory molecules. The underlying hypothesis proposes that stem cells’ bioactive molecules promote tendon regeneration by facilitating an early shift from the inflammatory phase to the proliferative healing phase. Based on this premise, the thesis aimed to produce AEC immune regulatory secretomes to use them as innovative stem cell-free solutions for controlling the early immune phase of tissue regeneration. The first step assessed whether the immunomodulatory properties of AEC depend on cell phenotype, considering their ability to undergo Epithelial-Mesenchymal Transition (EMT) to support regeneration or interact with different materials. In this context, we found that AEC promptly activated EMT when seeded on graphene oxide (GO)-coated surfaces, a carbon-based nanomaterial that has entered the field with several biomedical applications owing to its exceptional physicochemical and biological features. The cells interacted with GO by activating a specific TGF-β/SMAD signaling pathway that enhances some pro-regenerative AEC properties such as cell migration and adhesion but simultaneously diminishes their ability to inhibit immune cell activation (PBMC), thus confirming that when AEC shift toward the mesenchymal phenotype, their ability to modulate immune responses through paracrine signaling decreases. Subsequently, the study moved towards investigating the molecular mechanism that underlies the paracrine immunomodulatory properties that AEC can express both intrinsically and following exposure to external stimuli. AEC-derived amphiregulin (AREG)/EGFR axis was identified as a key intracellular pathway in regulating immune responses. The activation of this powerful immune-modulatory factor is controlled through the COX-2/PGE2/EP4 axis, which can be activated from different and sometimes converging pathways. First, LPS promotes it through NF-κβ activation but induces YAP degradation, while stretching promotes it through YAP activity. Combined, LPS suppresses stretching's effects, emphasizing the need to balance these stimuli for fine-tuning their anti-inflammatory effects. Beyond cellular responses, this thesis delved into the fractions of the AEC immune modulatory secretome. To this aim, microvesicles (MV) and MV-free fractions were isolated, characterized, and studied for their modulating immune function. It was found that AEC exert their immune modulatory activities using both MV-free and MV secretome fractions. For the first time, we demonstrated that the MV fraction of AEC secretome is enriched of organelle cargo (mitochondria, RE) that can be actively internalized in immune cells, triggering mitochondria-dependent apoptosis and thereby highlighting a potent, cell-free immune suppressive mechanism that could offer therapeutic advantages without the need for direct cell transplantation. Interestingly, the MV-free fraction of the AEC secretome was more effective in inhibiting PBMC and Jurkat cell activation in response to LPS stimuli by rapidly increasing its soluble immunomodulatory components. To translate these findings into potential clinical treatments, the AEC secretome was then encapsulated within a Hyaluronic Acid- Polyethylene Glycol-Heparin (HA-PEG-HEP) hydrogel matrix, which provided a controlled and sustained release of bioactive molecules, significantly bolstering both immunosuppressive and pro-regenerative effects over extended periods. This encapsulation strategy preserved the bioactivity of crucial components like AREG, advancing the therapeutic potential of AEC secretome in a practical, scalable form suitable for clinical use. By harnessing AEC's ability to modulate immune responses and support tissue repair, this dissertation lays the groundwork for the development of cell-free therapies that have broad implications for managing inflammation and degenerative conditions, marking a significant step toward translating AEC-based regenerative therapies from bench to bedside.","Jännetautien, joihin kuuluvat sekä rappeuttavat että tulehdukselliset jännekudoksen sairaudet, muodostavat merkittävän kliinisen haasteen, vaikuttaen miljooniin ihmisiin maailmanlaajuisesti ja aiheuttaen huomattavan sosioekonomisen taakan. Jännetautien kehittyminen on usein krooninen prosessi, jossa parantumisprosessi jää kesken, mikä johtaa immuunisolujen, kuten makrofagien, neutrofiilien ja lymfosyyttien, jatkuvaan aktivoitumiseen. Nämä solut vapauttavat tulehdusta edistäviä sytokiineja ja matriksia hajottavia entsyymejä. Pitkittynyt tulehdusvaihe, yhdistettynä jännekudoksen alhaiseen uusiutumiskykyyn, estää kudoksen korjaantumista ja johtaa epäjärjestäytyneeseen soluväliaineeseen, heikentäen kudoksen biomekaanisia ominaisuuksia. Nykyiset hoitokeinot lievittävät lähinnä oireita, mutta eivät kohdenna sairauden taustalla olevia tulehduksellisia mekanismeja, jotka aiheuttavat patologian ja lisäävät uusiutumisriskiä. Tämä väitöskirja tutkii amnionin epiteelisolujen (AEC) uusia pro-tenogeenisiä ominaisuuksia, jotka välittyvät ensisijaisesti niiden parakriinisten immunomodulatoristen molekyylien kautta. Perusolettamuksena on, että kantasolujen bioaktiiviset molekyylit edistävät jänteen uudistumista helpottamalla tulehdusvaiheen varhaista siirtymistä lisääntymisvaiheeseen. Tämän pohjalta väitöskirjan tavoitteena oli tuottaa AEC:n immunomodulatorisia eritystuotteita ja hyödyntää niitä uusina kantasoluttomina ratkaisuina kudoksen uudistumisen varhaisen immuunivaiheen hallintaan. Ensimmäinen vaihe oli arvioida, riippuuko AEC:n immunomodulatorinen potentiaali solujen fenotyypistä, kun otetaan huomioon niiden kyky käydä läpi epiteeli-mesenkymaali transitiota (EMT) kudoksen uusiutumisen tukemiseksi tai niiden vuorovaikutus eri materiaalien kanssa. Tässä yhteydessä havaitsimme, että AEC käynnistivät nopeasti EMT:n, kun ne kylvettiin grafeenioksidilla (GO) pinnoitetuille pinnoille – hiilipohjainen nanomateriaali, jolla on useita biolääketieteellisiä sovelluksia erinomaisen fysiokemiallisen ja biologisen ominaisuutensa ansiosta. Solut vuorovaikuttivat GO:n kanssa aktivoimalla TGF-β/SMAD-signaalireitin, joka parantaa tiettyjä AEC:n pro-regeneratiivisia ominaisuuksia, kuten solujen migraatiota ja adheesiota, mutta samanaikaisesti vähentää niiden kykyä estää immuunisolujen aktivoitumista (PBMC). Tämä vahvisti, että kun AEC siirtyvät mesenkymaaliseksi fenotyypiksi, niiden kyky säädellä immuunivastetta parakriinisen signaloinnin kautta heikkenee. Seuraavaksi tutkimuksessa selvitettiin molekyylimekanismeja, jotka säätelevät AEC:n parakriinista immunomodulatorista vaikutusta sekä luontaisesti että ulkoisten ärsykkeiden seurauksena. AEC:n erittämän amfireguliinin (AREG)/EGFR-akselin havaittiin olevan keskeinen immuunivasteiden säätelyssä. Tämän voimakkaan immunomodulatorisen tekijän aktivointi tapahtuu COX-2/PGE2/EP4-reitin kautta, joka voi aktivoitua useista eri ja joskus yhdistyvistä reiteistä. Esimerkiksi LPS edistää sen aktivaatiota NF-κB:n kautta mutta samalla hajottaa YAP:ia, kun taas venytys edistää YAP-aktiivisuutta. Yhdessä LPS estää venytyksen vaikutuksia, mikä korostaa tarvetta tasapainottaa näitä ärsykkeitä niiden tulehdusta estävien vaikutusten hienosäätämiseksi. Soluvastausten lisäksi tutkimuksessa keskityttiin AEC:n immunomodulatorisen eritystuotteen osajakeisiin. Tätä varten mikrovesikkelit (MV) ja MV-vapaat jakeet eristettiin, karakterisoitiin ja tutkittiin niiden immuunivasteita säätelevän vaikutuksen osalta. Havaittiin, että AEC toteuttavat immunomodulatoriset vaikutuksensa sekä MV-vapaan että MV-eritystuotejakeiden kautta. Ensimmäistä kertaa osoitettiin, että AEC:n eritystuotteiden MV-jakeet sisältävät organelleja (mitokondrioita, RE), jotka voivat aktiivisesti siirtyä immuunisoluihin, laukaisten mitokondrioriippuvaisen apoptoosin. Tämä paljasti voimakkaan, soluttoman immuunisuppressiivisen mekanismin, joka voi tarjota terapeuttisia etuja ilman suoraa solusiirtoa. Kiinnostavasti havaittiin, että MV-vapaa eritystuotejakso oli tehokkaampi PBMC:n ja Jurkat-solujen aktivaation estämisessä LPS-ärsykkeeseen vastaamalla, sillä se lisäsi nopeasti liukoisten immunomodulatoristen komponenttien määrää. Näiden löydösten kliinistä soveltamista varten AEC:n eritystuote kapseloitiin hyaluronihappo-polyetyleeniglykoli-hepariini (HA-PEG-HEP) -hydrogeelimatriksiin, joka tarjosi kontrolloidun ja pitkäkestoisen bioaktiivisten molekyylien vapautumisen, vahvistaen merkittävästi sekä immunosuppressiivisia että pro-regeneratiivisia vaikutuksia pitkällä aikavälillä. Tämä kapselointistrategia säilytti kriittisten komponenttien, kuten AREG:n, bioaktiivisuuden, edistäen AEC-eritystuotteiden terapeuttista potentiaalia kliinisessä, skaalautuvassa muodossa. Hyödyntämällä AEC:n kykyä säädellä immuunivasteita ja tukea kudoksen uusiutumista, tämä väitöskirja luo perustan soluttomille terapioille, joilla on laaja-alaisia vaikutuksia tulehduksellisten ja rappeuttavien sairauksien hallintaan. Tämä merkitsee merkittävää askelta kohti AEC-pohjaisten regeneratiivisten hoitojen viemistä laboratoriosta kliiniseen käyttöön."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10138/591573"],"dc:language.iso":["eng"],"dc:publisher":["University of Helsinki","Helsingin yliopisto","Helsingfors universitet"],"dc:rights":["This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.","Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.","Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden."],"dc:subject":["drug Research"],"dc:title":["Stem Cell-Based Immune Modulation and Regeneration"]},"updated_at":"2026-08-21T22:21:56Z"}