Morfološka i molekularna karakterizacija humanog masnog tkiva i lipoma različitih anatomskih lokalizacija i ispitivanje izolovanih mezenhimskih ćelija in vitro

Abstract

Lipomi su benigni tumori masnog tkiva sa još uvek nedovoljno razjašnjenom etiologijom i patogenezom. Lipomi su uglavnom lokalizovani potkožno ali se mogu naći i intramuskularno, u telesnim šupljinama i organima, mogu biti solitarni ili multipli, mogu varirati u veličini i histološkoj građi, ali podaci o njihovom molekularnom profilu i mehanizmima nastanka su vrlo oskudni. Mezenhimske matične ćelije iz lipoma (eng. lipomaderived stem cells - LDSCs) su predložene za primenu u tkivnom inženjerstvu i regenerativnoj medicini od strane autora nekoliko studija zato što je pokazano da imaju karakteristike slične mezenhimskim matičnim ćelijama iz masnog tkiva (eng. adipose-derived stem cells - ADSCs). Osim podataka o potencijalu izolovanih LDSCs da proliferišu i da se diferentuju u adipocite, osteoblaste i hondrocite, nema podataka o ostalim karakteristikama ovih ćelija a ni detaljnog poređenja sa ADSCs na ćelijskom i molekularnom nivou. Cilj jednog dela ovog istraživanja je bio ispitivanje molekularnog profila izolovanih LDSCs i poređenje sa ADSCs u kulturi in vitro, ispitivanje kapaciteta LDSCs da se diferentuju u adipocite i osteoblaste, na ćelijskom, proteinskom i genskom nivou, ispitivanje uticaja kondicioniranih medijuma LDSCs na odgovor makrofaga, kao i ispitivanje imunomodulacijske aktivnosti i efekta na zarastanje rana u sistemu indirektne ko-kulture in vitro, u poređenju sa ADSCs. Cilj drugog dela ovog istraživanja je bio morfološka i molekularna karakterizacija tkiva lipoma i potkožnog belog masnog tkiva (eng. subcutaneous white adipose tissue – scWAT) različitih anatomskih lokalizacija. Rezultati su pokazali da LDSCs i ADSCs poseduju karakteristike mezenhimskih matičnih ćelija i da imaju sličan fenotip ali različitu molekularnu osnovu za diferencijaciju. Obrazac ekspresije gena markera adipogeneze i prisustvo manjeg broja zrelih adipocita u kulturi LDSCs u odnosu na ADSCs nakon 21 dan adipogene diferencijacije ukazuju na slabiji kapacitet LDSCs za diferencijaciju u adipocite u poređenju sa ADSCs. Analiza osteogenih markera nakon 16 dana osteogene diferencijacije je pokazala da i LDSCs i ADSCs imaju fenotip karakterističan za osteoblaste, ali su bile u različitim fazama osteogeneze. Kondicionirani medijumi LDSCs (LDSC-CM) i ADSCs (ADSC-CM) dovode do funkcijske aktivacije nestimulisanih makrofaga i ispoljavaju anti-inflamacijsku aktivnost, sa izraženijim efektom LDSC-CM. LDSC-CM i ADSC-CM deluju povoljno na zarastanje rana u sistemu indirektne ko-kulture in vitro, bez značajnih razlika, i u nivou su pozitivne kontrole...

Lipomas are benign adipose tissue tumors with still insufficiently clarified etiology and pathogenesis. Lipomas are mainly localized subcutaneously but can also be found intramuscularly, in the body cavities and organs, may be solitary or multiple, may vary in size and histological structure, but data on their molecular profile and mechanisms of formation are still scarce. Lipoma-derived stem cells (LDSCs) have been proposed for use in tissue engineering and regenerative medicine by the authors of several studies since it has been shown that these cells have characteristics similar to adipose-derived stem cells (ADSCs). In addition to data on the potential of isolated LDSCs to proliferate and differentiate into adipocytes, osteoblasts and chondrocytes, there is no data on other characteristics of these cells, nor a detailed comparison with ADSCs at the cellular and molecular levels. The aim of one part of this study was to examine the molecular profile of isolated LDSCs and comparison with ADSCs in culture in vitro, to examine the capacity of LDSCs to differentiate into adipocytes and osteoblasts on cellular, protein and gene level, to examine the influence of conditioned media of LDSCs on macrophages’ response, as well as to examine immunomodulatory activity and effect of wound healing in indirect coculture system in vitro, in comparison with ADSCs. The aim of the second part of this study was the morphological and molecular characterization of lipoma tissue and subcutaneous white adipose tissue (scWAT) of various anatomical localizations. The results showed that LDSCs and ADSCs possess the characteristics of mesenchymal stem cells and have similar phenotype but different molecular basis for differentiation. The pattern of expression of genes that are markers of adipogenesis and the presence of less number of mature adipocytes in LDSCs compared to ADSCs culture after 21 day of adipogenic differentiation, indicate lower capacity of LDSCs for differentiation into adipocytes compared to ADSCs. Analysis of osteogenic markers after 16 days of osteogenic differentiation showed that both LDSCs and ADSCs have a characteristic osteoblast-like phenotype, but they were at different stages of osteogenesis...