Date of Award

11-1-2024

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Hammad Hassan

Second Advisor

Dr. Afsar Ali Mian

Department

Biological and Biomedical Sciences

Abstract

Blood transfusion plays a critical role in healthcare, especially in low- and middle-income countries (LMICs), according to a World Health Organization (WHO) report over 54% of transfusions are for children under five, and 76% for patients over age 60. Donation rates are lower in LMICs (6.6%) compared to high-income countries (31.5%), with the lowest at 5% in low-income nations. This trend, coupled with a decreasing blood supply and increasing demand, will exacerbate shortages in LMICs. In Pakistan, blood transfusion services are disputed by disorganization and quality issues, with reliance on 170 public and 450 private blood banks. Currently, these services meet only 60% of the annual demand for 1.5 million units, resulting in a 40% shortfall. This heightens the risk of alloimmunization and complicates donor compatibility. Patients with rare blood types or chronic conditions are particularly vulnerable, facing increased risks of transfusion reactions and infections like hepatitis B and HIV.
Objective: This research study aims to utilize hematopoietic stem cells (HSCs) derived from cord blood to differentiate into erythroid cells, focusing on generating and characterizing an immortalized adult erythroid cell line, and driving its differentiation towards red blood cell (RBC) production.
Methods Cord blood samples were taken from consenting pregnant mothers, and CD34+ HSCs were isolated and cultured to analyse differentiation over a 14-day period in controlled media. Cell counting and technique like Fluorescence activated cell sorting were performed at four different key time points to observe the cell surface markers progression. A lentiviral vector containing HPV16 E6/E7 genes was used to infect CD34+ cells. Mononuclear cells (MNCs) were cultured in two different media formulations from Day 0 to Day 7, then transitioned to tertiary media from Day 7 to Day 14 to promote RBC differentiation. Giemsa staining with the help of methylene blue dye helped in confirmation of morphological changes and the FACS analysis assisted in tracking the expression of cell surface markers CD34, CD45, CD71 and CD235a to assess the differentiation process.
Results MNCs from patient-derived blood differentiated into erythroid cells over 14 days using controlled Media I and Media II. Phase contrast microscopy and FACS analysis confirmed differentiation, with surface markers showing a major shift at Day 4, including a decrease in CD34 and peak formation in CD71 expression. By Day 14, cell progression, in the erythropoiesis differentiation stages, visible viii formation of basophilic, polychromatic and orthochromatic cells could be seen. FACS analysis indicated a decline in CD34 and CD45 expression from Day 4, while CD71 and CD235a markers increased significantly by Day 14, reflecting the maturation of MNCs into RBC precursors. Peaks in CD71 (80.1%) and CD235a (18.6%) on Day 14 confirmed successful erythroid differentiation.
Conclusion This study successfully generated erythroid progenitor lines from cord blood by advancing MNCs to the orthochromatic normoblast stage using growth factors such as EPO, SCF, IL-3, FLT-3, and TPO. Differentiation of MNCs towards RBC maturation was validated by FACS and confirmed by Giemsa staining, highlighting the significant changes in the morphology and surface markers of cells over time (CD34, CD45, CD71, CD235a). Statistical analyses using R software confirmed the normality and sphericity of the data. The statistical Friedman test indicated a significant difference in the cell surface markers expression across different time points, and a Linear Mixed Model (LMM) demonstrated a strong relationship between CD71 expression and cell differentiation, with an R-squared value of 92.85%. Post hoc comparisons showed CD71’s highest average expression, emphasizing its importance in the differentiation process. Overall, the findings replicate natural erythropoiesis under controlled conditions and provide foundational insights for developing therapies for RBC-related disorders. These results answer the research question by demonstrating that, cord blood derived CD34+ cells can be successfully immortalized and differentiated into erythroid cell lineage , offering a scalable model for therapeutic application.

First Page

1

Last Page

98

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