Characterization of Stem Cell Phenotype and Adipogenic Potential of Human and Rat Primary Adipose-Derived Mesenchymal Stem Cells
Author
Okdahl, Tina
Term
4. term
Publication year
2018
Submitted on
2018-05-30
Pages
50
Abstract
Baggrund: Heling af hudsår er en kompleks proces, som kan gå i stå, hvis der er for mange betændelsesfremmende faktorer i vævet. Kroniske sår, der forbliver i en langvarig betændelsestilstand, er et voksende sundhedsproblem. Mesenkymale stamceller (MSC’er) er stamceller fra voksent væv, som kan dæmpe betændelse, især via parakrine mekanismer – det vil sige ved at udskille signalstoffer, der påvirker naboceller. Derfor er det interessant at undersøge, om MSC’er kan hjælpe med at skifte et sår fra en betændelsespræget til en mere helingsfremmende tilstand. Formål: At karakterisere stamcelleegenskaberne hos humane og rotte adipose-afledte mesenkymale stamceller (hASC’er og rASC’er) og undersøge deres evne til at danne fedt, samt at vurdere deres vækst, klondannelse og om de udskilte stoffer kan fremme dannelse af nye blodkar (angiogenese) i celleforsøg. Metoder: Primære ASCs blev isoleret fra menneskers og rottes bugfedt og dyrket i medier, der skulle fremme udvikling til fedt-, knogle- og bruskceller. Differentiering blev vurderet både ved farvning af celler og på molekylært niveau. Vi målte også cellernes delingshastighed og deres evne til at danne kolonier fra enkelte celler. Endelig blev der udført såkaldte endothelial scratch assays – et laboratorieforsøg, hvor et lag af blodkarceller ridses, så man kan måle, hvor hurtigt “såret” lukker – for at vurdere den angiogene effekt af konditioneret medie (dyrkningsvæsken) fra hASC- og rASC-kulturer. Resultater: I humane ASC-kulturer lykkedes det at inducere fedt- og bruskdannelse, mens der i rottekulturer kun blev opnået fedtdannelse. Både hASC’er og rASC’er viste høj vækstkapacitet og kunne danne kolonier. Konditioneret medie fra hASC-kulturer fremskyndede ikke helingen i scratch-forsøget. Omvendt forkortede konditioneret medie fra rASC’er signifikant helingstiden for et lag af rotte-endotelceller. Konklusion: De isolerede hASC’er og rASC’er havde de forventede egenskaber for mesenkymale stamceller, men yderligere forsøg med differentiering til fedt, knogle og brusk er nødvendige for endeligt at bekræfte multipotens. At rASC-mediet reducerede helingstiden tyder på, at rASC’er udskiller faktorer, der fremmer dannelse af blodkar, hvilket er relevant for fremtidige studier i en diabetisk rotte-model for kroniske sår.
Background: Skin wound healing is a complex process that can stall when too many pro‑inflammatory signals are present. Chronic wounds that remain locked in inflammation are a growing healthcare problem. Mesenchymal stem cells (MSCs) are adult stem cells that can dampen inflammation, mainly through paracrine mechanisms—by releasing signals that act on neighboring cells. This raises the possibility of using MSCs to shift a chronic wound from an inflammatory to a more healing‑friendly state. Aim: To characterize the stem cell phenotype of human and rat adipose‑derived mesenchymal stem cells (hASCs and rASCs) and assess their ability to become fat cells, as well as their growth, colony formation, and whether the substances they secrete can promote new blood vessel growth (angiogenesis) in cell‑based tests. Methods: Primary ASCs were isolated from human and rat abdominal fat and cultured in media intended to induce differentiation into fat, bone, and cartilage. Differentiation was evaluated by histochemical staining and at the molecular level. We also measured proliferation rates and the fraction of cells capable of forming colonies from single cells. Finally, endothelial scratch assays—where a “wound” is created in a layer of blood vessel lining cells to track closure over time—were used to test the angiogenic effects of conditioned medium (the culture fluid collected from cell cultures) from hASCs and rASCs. Results: In human ASC cultures, adipogenic (fat) and chondrogenic (cartilage) differentiation were achieved, whereas only adipogenic differentiation was obtained in rat cultures. Both hASCs and rASCs showed high proliferative capacity and clonogenicity. Conditioned medium from hASCs did not accelerate wound closure in the in vitro scratch assay. In contrast, conditioned medium from rASCs significantly shortened the healing time of a rat endothelial cell monolayer. Conclusion: The phenotypic analyses indicate that the isolated hASCs and rASCs behaved as active, viable mesenchymal stem cells, but further tri‑lineage differentiation (fat, bone, cartilage) is needed to confirm multipotency. The faster closure seen with rASC‑conditioned medium suggests that rASCs secrete factors that support angiogenesis, which is relevant for future studies in a diabetic rat model of chronic wounds.
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