Development of screening platforms for enhancing production and evaluation of platelets produced in vitro

Viraj Kamat, University of Pennsylvania


Platelets produced in vitro from stem cells have the potential to serve as a supplementary source for clinical transfusions. Platelet production in an adult human is an extremely efficient process, generating approximately 1011 platelets per day. Current culture methods have not yet been able to produce platelets in sufficient quantities required for transfusion. Additionally, several studies have shown that culture-derived platelets have reduced functionality compared to blood platelets. This work presents the development of screening platforms to enhance the production and functional evaluation of culture-derived platelets. In the first portion, a 96-well microRNA (miRNA) screen was developed to identify miRNAs that regulate the differentiation of embryonic stem cell derived hematopoietic progenitors. Four-color high throughput flow cytometry was used to assess differentiation towards megakaryocyte, erythroid or myeloid lineages following miRNA transfection. The screen revealed miR-105 as a novel enhancer of the megakaryocyte lineage. We further showed that miR-105 promotes megakaryopoiesis from adult CD34+ cells. Finally, we demonstrated that miR-105 regulated megakaryopoiesis in part by targeting the hematopoietic transcription factor c-myb. In the second portion, a microfluidic assay was developed to assess the functional incorporation of culture-derived platelets (CDPs) into a growing human thrombus under flow. Our method required significantly lesser CDPs compared to in vivo incorporation assays in mice. CDPs incorporated into clots at reduced efficiency of blood platelets, though both cells types were equally inhibited by αIIbβ3 antagonist. Finally, the assay could monitor the incorporation of CDPs produced from transiently transfected megakaryocytes, thereby demonstrating the potential of assessing genetically modified platelets.

Subject Area

Molecular biology|Cellular biology|Chemical engineering|Oncology

Recommended Citation

Kamat, Viraj, "Development of screening platforms for enhancing production and evaluation of platelets produced in vitro" (2015). Dissertations available from ProQuest. AAI3709488.