Optimizacija večbarvne pretočne citometrije za imunofenotipizacijo človeških primarnih mezenhimskih matičnih/stromalnih celic

Selan Podpečan, Nina (author), Zupan, Janja (mentor)

Mezenhimske matične/stromalne celice (MSC) predstavljajo heterogeno populacijo celic, ki se v mirujočem, nediferenciranem stanju nahaja v številnih vezivnih tkivih. Njihova zanesljiva identifikacija je ključna za raziskave in uporabo v regenerativni medicini. Ker specifičen označevalec ne obstaja, je za njihovo identifikacijo potrebno analizirati nabor pozitivnih in negativnih celičnih označevalcev. Večbarvna pretočna citometrija omogoča takšno natančno imunofenotipizacijo MSC, vendar zahteva skrbno načrtovanje in optimizacijo panela. Namen magistrskega dela je bila optimizacija panela za večbarvno pretočno citometrijo za imunofenotipizacijo primarnih humanih MSC, s poudarkom na celicah iz perinatalnih tkiv. Na podlagi smernic Mednarodnega združenja za celično in gensko terapijo (angl. International Society for Cell and Gene Therapy; ISCT) in pregleda literature smo izbrali ustrezne negativne (CD45, CD235a, CD146) in pozitivne označevalce (CD73, CD105, CD90) ter dodatna označevalca PDPN in CD164 za opredelitev nove populacije humanih skeletnih matičnih celic (angl. human skeletal stem cells, hSSC). Panel smo optimizirali s postopnim vključevanjem protiteles, določitvijo optimalnih volumnov ter prilagoditvijo nastavitev pretočnega citometra. Uspešnost optimizacije panela smo preverili s kontrolami s selektivno izločenim posameznim fluorokromom (angl. fluorescence minus one, FMO), ki so nam bile v pomoč tudi pri določitvi ustreznih mej med pozitivnimi in negativnimi populacijami. Rezultate optimizacije smo sproti analizirali in iskali možnosti za izboljšave v panelu. Analiza kontrol FMO nam je dala boljši vpogled v oceno ozadja fluorescence in potencialnega spektralnega prekrivanja ter širjenja signala. Uporabnost optimiziranega panela smo preverili na vzorcih primarnih celic pridobljenih iz placente, kjer smo z imunofenotipizacijo določili deleže preiskovanih celičnih populacij. Rezultati so pokazali visoko izražanje CD73 ter variabilno izražanje CD105 in CD90, kar odraža heterogenost MSC populacij, medtem ko je izražanje PDPN nakazovalo na prisotnost subpopulacij MSC. Optimiziran panel predstavlja uporabno metodo za imunofenotipizacijo MSC s standardnimi in novejšimi označevalci ter pomembno osnovo za nadaljnje raziskave na področju matičnih celic. Hkrati je panel pomembna osnova, ki dopušča nadaljnje izboljšave in nadgradnjo potencialnih novih označevalcev.

• Language: Slovenian
• Keywords: Ključne besede: mezenhimske matične/stromalne celice (MSC), večbarvna pretočna citometrija, imunofenotipizacija, optimizacija, perinatalne MSC
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Publisher: [N. Selan Podpečan]
• Year: 2026
• PID: 20.500.12556/RUL-183396
• UDC: 602.9(043.2)
• COBISS.SI-ID: 281587203
• Publication date in RUL: 12.06.2026

Secondary language

• Language: English
• Title: Optimization of multi-colour flow cytometry for immunophenotyping of human primary mesenchymal stem/stromal cells

Abstract

Mesenchymal stem/stromal cells (MSCs) represent a heterogenous population of cells residing in a quiescent, undifferentiated state across various connective tissues. Their reliable identification is essential for both research and applications in regenerative medicine. As no single specific marker exists, their identification requires the analysis of a combination of positive and negative cellular markers. Multicolour flow cytometry enables precise immunophenotyping of MSCs, however, it requires careful panel design and optimization. The aim of this master's thesis was to optimize a multicolour flow cytometry panel for immunophenotyping of primary human MSCs, with a focus on cells derived from perinatal tissues. Based on the guidelines of the International Society for Cell and Gene Therapy (ISCT) and a review of the literature, we selected appropriate negative (CD45, CD235a, CD146) and positive markers (CD73, CD105, CD90), as well as additional markers PDPN and CD164 to define a novel population of human skeletal stem cells (hSSCs). We optimized the panel through the stepwise inclusion of antibodies, determination of optimal reagent volumes, and adjustment of flow cytometer settings. The performance of the optimized panel was evaluated using fluorescence minus one (FMO) controls, which also aided in establishing accurate gating between positive and negative populations. The optimization results were continuously analyzed to identify opportunities for improvement. Analysis of FMO controls provided deeper insight into background fluorescence, as well as potential spectral overlap and spreading. The applicability of the optimized panel was further tested on primary cell samples derived from the placenta, where immunophenotyping was used to determine the proportions of the investigated cell populations. The results demonstrated high expression of CD73 and variable expression of CD105 and CD90, reflecting the heterogeneity of MSC populations, while PDPN expression indicated the presence of MSC subpopulations. The optimized panel represents a useful method for MSC immunophenotyping, incorporating both established and emerging markers, and provides a solid foundation for further research in the field of stem cells. At the same time, it offers flexibility for future improvements and the integration of potential new markers.

• Keywords: Keywords: mesenchymal stem/stromal cells (MSCs), multicolour flow cytometry, immunophenotyping, optimization, perinatal MSCs