Obtinerea de celule cu caracter cardiomiocitar prin transdiferentierea celulelor mezenchimale umane derivate din maduva osoasa hematogena

    Autori: Dan Marian Danila, Alin Lucian Popescu, Virgil Paunescu

Scopul studiului de fata consta in prelucrarea in vitro a celulelor mezenchimale umane derivate din maduva osoasa hematogena umana prin procesul de transdiferentiere spre celule cu caracter cardiomiocitar. Ca agent de transdiferentiere, am utilizat 5-azacitidina, alaturi de amphotericina, Tyrode’s balanced salt solution, bFGF intr-un mod unic de utilizare prin expunere unica de 24 de ore la 5-azacitidina, urmata in continuare de expunere la mediu complet fara 5-azacitidina pentru o saptamana, doua respectiv trei saptamani pana la terminarea experimentului. Celulele obtinute au fost analizate prin microscopie optica, electronica, imunohistochimie si prin prezenta markerilor de biologie moleculara prin RT-PCR. În microscopia optica, la o saptamana dupa transdiferentiere, aproximativ 20% din celulele ramase viabile au dobandit caracter fusiform, asemanator celulelor fibroblastice, la 2 saptamani, un procent 60% au avut caractere similare dintre celulele viabile, iar la 3 saptamani, aproximativ 80% dintre celulele viabile au prezentat aceste caracteristici. Microscopia electronica a relevat faptul ca celulele mezenchimale transdiferentiate viabile la 3 saptamani au format miofilamente orientate paralel la nivelul citoplasmei, fara insa a fi unite in sarcomere. Acest lucru nu a fost observat la experimentele terminate la una, respectiv doua saptamani. Aceste miofilamente nu au fost de asemenea observate la analiza celulelor nediferentiate. La 3 saptamani dupa transdiferentiere, aproximativ 60% din celulele transdiferentiate au prezentat reactie pozitiva pentru ßmyosin heavy chain, desmina, si -actinina la analiza imunohistochimica comparativ cu lotul de control. Analiza de RT-PCR a aratat expresie crescuta a 4 markeri specifici cardiomiocitari (ß-myosin heavy chain, Tropomiozina, -actinina, Troponina I) in celulele transdiferentiate la 3 saptamani comparativ cu lotul de control. În concluzie, transdiferentierea in vitro a celulelor mezechimale umane derivate din maduva osoasa hematogena in celule cu caracter cardiomiocitar poate fi realizata cu succes cu ajutorul 5-azacitidinei, conform metodei descrise de noi, celulele derivate prezentand markeri caracteristici cardiomiocitari confirmati prin imunohistochimie si biologie moleculara.

Cuvinte cheie: celule mezechimale, cardiomiocite, transdiferentiere, 5-azacitidina.

The goal of the current study consists of transdifferentiang the human mesenchymal stem cells derived from the bone marrow into cardiomyocytelike cells. Materials and methods: As transdifferentiating agent we used 5-azacytidine, along with a mixture of amphotericin, Tyrode’s balanced salt solution, bFGF used in an unique way by exposing the cells to 5-azacytidine for 24h only, followed by continous cell maintenance medium without 5-azacytidine for one, two and respectively 3 weeks until the end of the experiment. The derived cells were analysed by optic and electronic microscopy, imunohistochemistry and RT-PCR. Results: One week after transdifferentiation, around 20% of the viable cells had a fusiform appearance seen in optic microscopy, similar to the one of the fibroblastic cells; at 2 weeks around 60% had this characteristic and 80% had this characteristic at 3 weeks. In electronic microscopy, the transdifferentiated cells showed parallel miofilaments inside the cytoplasm, which didn’t form sarcomeric structures though. This was not seen in the experiments finished at one and two weeks respectively. These miofilaments were not seen also in the undifferentiated cells which proves the miogenic transdifferentiation. At 3 weeks from the transdifferentiation treatment, around 60% of transdifferentiated cells were positive for ß-myosin heavy chain, desmin, and -actinin on imunohistochemical staining compared to the undifferentiated group. RT-PCR analysis showed statistically significant increased expression of ß-myosin heavy chain, tropomiozin, -actinin, troponin I at 3 weeks after transdifferentiation compared to the undifferentiated, control group. Conclusion: the transdifferentiation in vitro of bone marrow derived mesenchymal stem cells into cardiomyocyte-like cells can be successfully done with 5-azacytidine according to the method described above, the derived cells displaying cardiomyocyte markers by imunohistochemistry and molecular biology analysisKey words: premature spontaneous birth, transvaginal scan, cervical length assesment.

Key words: mesenchymal stem cells, cardiomyocytes, transdifferentiation, 5-azacytidine. 

Departamentul de Fiziologie-Imunologie, Universitatea de Medicina si Farmacie “Victor Babes”, Timisoara

Adresa pentru corespondenta   Email: This e-mail address is being protected from spam bots, you need JavaScript enabled to view it