Date of Award

10-2025

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Muhammad Zuhair Yusuf

Second Advisor

Dr. Hammad Hassan

Third Advisor

Dr Rehana Rehman

Department

Biological and Biomedical Sciences

Abstract

Background Platelets are vital for hemostasis, tissue repair, and immune regulation. Their application in transfusion medicine is limited due to donor scarcity, short shelf life, and the risk of infection transmission. Megakaryocytes (MKs) are precursor cells that generate platelets, provide a valuable cellular source for in-vitro platelet production. By generating MKs from CD34+ HSCs, donor reliance can be avoided while advancing thrombopoiesis knowledge. However, primary CD34+ HSCs' limited lifespan and restricted proliferation remain a significant challenge. This study aimed to design a protocol for the in-vitro MKs generation from CD34⁺ HSCs derived from cord blood (CB) and to assess lentiviral-driven immortalization as a potential strategy to extend HSC lifespan to provide sustainable and scalable sources of platelet generation for medical applications.
Methods Umbilical cord blood samples were collected in CPD anticoagulant tubes. Mononuclear cells (MNCs) were obtained through Ficoll-Paque density-gradient centrifugation, followed by Magnetic-Activated Cell Sorting (MACS) to isolate purified CD34⁺ HSC population. Isolated CD34+ HSCs cultured in Expansion medium supplemented with cytokines and growth factors and then transfer in MK-specific differentiation medium to generate mature megakaryocytes. Cell count and morphological changes were assessed by light microscopy and Giemsa staining, whereas immunophenotyping evaluation was conducted via flow cytometry analysis (FACS). Lentiviral system containing the HPV E6/E7 plasmid under doxycycline induction were produced in HEK 293T cells for the immortalization of CD34⁺ HSCs. 
 Results The combination of Ficoll gradient density centrifugation and MACS yield a significantly enriched CD34⁺ HSC population. Cultured CD34⁺ HSCs displayed progressive expansion and effective differentiation into mature megakaryocyte. Examination of MKs morphology via light microscopy and Giemsa staining, showed increased cell size, abundant cytoplasm, and irregular and lobulated nuclei. Whereas, polyploidy count and flowcytometry analysis of size and granularity, and immunophenotyping of MKs-specific surface markers were performed, shows successful generation of MKs. However, immortalization of CD34+ HSCs using lentiviral system is still in stage of optimization.
Conclusion This study successfully developed a reliable in-vitro model for generating megakaryocytes from CB–derived CD34⁺ HSCs, illustrating effective isolation, expansion and differentiation of CD34+ HSCs into megakaryocytes. While lentiviral immortalization is still in optimizing stage, these findings provide a solid foundation for future optimization of HSC immortalization, ultimately aiding in creation of scalable, donor-independent platelet production systems.

First Page

1

Last Page

79

Download

Share

COinS