Super-Blutplättchen als spezifische Transporter zur Regeneration des Herzens
Hämatologie, Onkologie
Zusammenfassung der Projektergebnisse
During this fellowship, it was intended to develop a new generation of human platelets produced in vitro which would allow a continuous, donor-independent supply for transfusion medicine. On the one hand, these platelets should be ‘universal’ which means they would be compatible with any recipient. On the other hand, their α-granules should be packed with beneficial proteins to improve for instance the regenerative capacity of different organs such as the heart. Therewith it would be possible to provide platelets designed specifically for the patients need at any time. Regarding the first part, we were able to genetically modify human pluripotent stem cell lines (ESCs as well as iPSCs) and eliminate HLA class I surface expression. These lines showed typical characteristics of stem cells and could be differentiated into megakaryocytes using a laboratory internal developed approach – called forward programming (FoP). Here, lentiviral particles (LVPs) provide three transcription factors GATA1, TAL1 and FLI1 mediating the differentiation. The HLA-null megakaryocytes produced by FoP showed phenotypic marker expression as well as other characteristic of Mks such as polyploidy and pro-platelet formation. Subsequently, they were able to produce viable HLA-null platelets. However, the produced amount of Mks and platelets was very variable due to the used LVPs. Therefore, the differentiation protocol was changed and replaced by an inducible TET-ON 3rdG approach. Here, genetically modified hPSC lines produce the needed transcription factors after doxycycline administration (inducible FoP). These hPSC lines were used to generate again HLA-null mutants. Characterization of the cells revealed that the stem cell phenotype is retained and differentiation into Mks resulted in high amounts of matured megakaryocytes and viable HLA-null platelets. In addition, up-scaling the Mk production would be fairly easy and cheap due to the inducible FoP. Trying to prove the superior survival of HLA-null Mks and platelets under refractory conditions in vivo proved to be challenging. So far no working protocol could be established. Most likely this is due to the used HLA-ABC antibody which seems to be unsuitable for the used mouse models. Currently, the experiments are repeated with another HLA-antibody. Regarding the second part, we were able to package proteins (eGFP) specifically into αgranules of Mks and platelets. This was done by using LVPs which contain a special construct encoding for a CD41 promotor fragment (allowing expression only in Mks), a targeting moiety (VWFSPD2) and eGFP. However, trying to insert the construct into the genomic safe harbour AAVS1 of hPSC lines to produce Mks with eGFP exclusively packed into α-granules turned out to be a challenge. At the moment, it appears that the GSH-AAVS1 is silenced during FoP and two new strategies are now tested. Either insulators will be added to the construct or another safe harbour – most likely the human ROSA26 locus – will be used. Establishing a mouse model for acute myocardial infarction with human platelets was also very challengin. Although human platelets accumulated in the MI area, only one NSG mouse out of five survived the procedure. At the minute, the MI protocol is revised by our collaboration partners to make it suitable for the more fragile NSG mice and to guaranty a better survival rate.
Projektbezogene Publikationen (Auswahl)
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(2019). Using genome editing to engineer universal Platelets. Emerg Top Life Sci. 3 (3): 301-311
Lawrence M, Mueller A, Ghevaert C
