By Maya Kardouh (Class of 2021), Julia Aguade Gorgorio, Iman Fares, and Hanna Mikkola
Leukemia, sickle cell anemia, beta-thalassemia, and other blood disorders can be cured by editing one’s defective Hematopoietic Stem Cells (HSCs) ex vivo and transplanting them back in vivo. However, because HSCs lose their “stemness” in culture, the genetic manipulation approach is not optimal. Because the regulatory mechanisms of HSCs are yet to be understood, the inability to maintain HSCs in culture compromises their therapeutic use in curing blood disorders (Magnusson, 2013). One of the genes The Mikkola lab found to be down-regulated in cultured human HSCs is MYCT1. The Mikkola lab had also determined that knockdown of MYCT1 compromised the engraftment of human HSCs in immunodeficient mice. Thus, it will be important to determine how is the MYCT1 protein involved in HSCs transplantability or engraftment. As there is minimal knowledge of how the MYCT1 protein functions, first, we have to determine where the MYCT1 protein is localized in HSCs, the protein domains responsible for its localization, and what protein complexes it interacts with. Using cell fractionation, immunofluorescence, co-immunoprecipitation, and Mass Spectrometry, we will answer these questions for the MYCT1 protein.
Because the number of HSCs available for research is limited, we carried out these experiments on cell lines first. We will perform the same experiments on HSCs after we have optimized our techniques on cell lines. So far, we have determined the localization of MYCT1 to be mainly in the membrane in cell line ACC697 (acute megakaryoblastic leukemia). Additionally, we identified the extracellular domain of MYCT1 as a director for its localization in the cell membrane. If we determine the localization of MYCT1 in HSCs to be the same as in cell line ACC697, we will turn next to question the biological role of MYCT1. Interestingly, endothelial cells express MYCT1 and were shown by the Mikkola lab to require MYCT1 for viability. If immunofluorescence and cell fractionation in endothelial cells and HSCs also show main localization of their MYCT1 in the cell membrane, then interactions of MYCT1 between HSCs and endothelial cells can be questioned. In the homing process in bone marrow, HSCs require integrins for adhesion and transendothelial migration (Sahin and Buitenhuis, 2012). Given that MYCT1 is a transmembrane protein and expressed both in HSCs and endothelial cells, it can be potentially required for adhesion during transendothelial migration. Thus, adhesion and transendothelial migration assays would be appropriate to test this hypothesis.
Determining MYCT1 localization and the protein complexes it actively interacts with, may reveal other proteins which silence it in culture. As determined by the Mikkola Lab, MYCT1 is downregulated in culture, and necessary but not sufficient for rescuing HSC function in vitro or in vivo. In parallel, the Mikkola lab has also identified MLLT3, another gene in HSCs to be down-regulated in culture and necessary for their maintenance. MLLT3 rescue expanded transplantable HSCs to 12-fold, but it did not rescue MYCT1. This suggests that inducing both MLLT3, and MYCT1 may facilitate more effective in vitro gene editing of HSCs, which can be used for transplantation and disease treatments.
References:
Magnusson, Mattias et al. “Expansion on stromal cells preserves the undifferentiated state of
human hematopoietic stem cells despite compromised reconstitution ability.” PloS one vol. 8,1(2013): e53912. doi:10.1371/journal.pone.0053912
Sahin, Aysegul Ocal, and Miranda Buitenhuis. “Molecular mechanisms underlying adhesion
and migration of hematopoietic stem cells.” Cell adhesion & migration vol. 6,1 (2012): 39-48. doi:10.4161/cam.18975
Picture from https://fineartamerica.com/featured/5-white-blood-cells-alfred-pasieka.html
Because the number of HSCs available for research is limited, we carried out these experiments on cell lines first. We will perform the same experiments on HSCs after we have optimized our techniques on cell lines. So far, we have determined the localization of MYCT1 to be mainly in the membrane in cell line ACC697 (acute megakaryoblastic leukemia). Additionally, we identified the extracellular domain of MYCT1 as a director for its localization in the cell membrane. If we determine the localization of MYCT1 in HSCs to be the same as in cell line ACC697, we will turn next to question the biological role of MYCT1. Interestingly, endothelial cells express MYCT1 and were shown by the Mikkola lab to require MYCT1 for viability. If immunofluorescence and cell fractionation in endothelial cells and HSCs also show main localization of their MYCT1 in the cell membrane, then interactions of MYCT1 between HSCs and endothelial cells can be questioned. In the homing process in bone marrow, HSCs require integrins for adhesion and transendothelial migration (Sahin and Buitenhuis, 2012). Given that MYCT1 is a transmembrane protein and expressed both in HSCs and endothelial cells, it can be potentially required for adhesion during transendothelial migration. Thus, adhesion and transendothelial migration assays would be appropriate to test this hypothesis.
Determining MYCT1 localization and the protein complexes it actively interacts with, may reveal other proteins which silence it in culture. As determined by the Mikkola Lab, MYCT1 is downregulated in culture, and necessary but not sufficient for rescuing HSC function in vitro or in vivo. In parallel, the Mikkola lab has also identified MLLT3, another gene in HSCs to be down-regulated in culture and necessary for their maintenance. MLLT3 rescue expanded transplantable HSCs to 12-fold, but it did not rescue MYCT1. This suggests that inducing both MLLT3, and MYCT1 may facilitate more effective in vitro gene editing of HSCs, which can be used for transplantation and disease treatments.
References:
Magnusson, Mattias et al. “Expansion on stromal cells preserves the undifferentiated state of
human hematopoietic stem cells despite compromised reconstitution ability.” PloS one vol. 8,1(2013): e53912. doi:10.1371/journal.pone.0053912
Sahin, Aysegul Ocal, and Miranda Buitenhuis. “Molecular mechanisms underlying adhesion
and migration of hematopoietic stem cells.” Cell adhesion & migration vol. 6,1 (2012): 39-48. doi:10.4161/cam.18975
Picture from https://fineartamerica.com/featured/5-white-blood-cells-alfred-pasieka.html
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