An In Vitro Assay : To Evaluate the Functional Roles of Peroxisomes in Normal Hematopoiesis
(2024) KIMM01 20241Educational programmes, LTH
Department of Immunotechnology
- Abstract
- Hematopoiesis refers to the formation of mature blood cells, a process driven by hematopoietic stem cells (HSCs) in the bone marrow. These rare cells are characterized by their long-term self-renewal and multilineage differentiation potential, enabling the maintenance and reconstitution of the hematopoietic system. Reactive oxygen species (ROS) are key regulators of normal hematopoiesis, and peroxisomes play a crucial role in ROS production and catabolism, yet their role in hematopoiesis is underexplored. This study establishes an in vitro assay to evaluate the functional roles of peroxisomes in hematopoiesis. The assay employs flow cytometry to precisely identify, analyze, and isolate HSCs. The CRISPRi guide RNA (gRNA) library and a... (More)
- Hematopoiesis refers to the formation of mature blood cells, a process driven by hematopoietic stem cells (HSCs) in the bone marrow. These rare cells are characterized by their long-term self-renewal and multilineage differentiation potential, enabling the maintenance and reconstitution of the hematopoietic system. Reactive oxygen species (ROS) are key regulators of normal hematopoiesis, and peroxisomes play a crucial role in ROS production and catabolism, yet their role in hematopoiesis is underexplored. This study establishes an in vitro assay to evaluate the functional roles of peroxisomes in hematopoiesis. The assay employs flow cytometry to precisely identify, analyze, and isolate HSCs. The CRISPRi guide RNA (gRNA) library and a Doxycycline-inducible CRISPR/dCas9-KRAB system enable specific manipulation of peroxisomal gene expression. A polyvinyl alcohol (PVA)-based culture system facilitates the in vitro expansion of HSCs, and gRNA delivery via lentiviral transduction. The functional roles of peroxisomes in HSC maintenance and differentiation are assessed using different in vitro culture systems. Through this workflow, PEX genes, which encode proteins essential for peroxisome function, can be analyzed to draw initial conclusions about their roles in normal hematopoiesis. To further evaluate the stem cell activity of candidate HSCs in vivo, bone marrow transplantation into conditioned (irradiated) wild-type recipients is required. Identifying peroxisomes as a novel metabolic factor in HSC biology could provide a strategy to enhance HSC function through the pharmacological targeting of peroxisome metabolism, offering potential advances to counteract hematological diseases. (Less)
- Popular Abstract
- Hematopoiesis is an important biological process that keeps you alive and healthy. This process produces different blood cells, such as red blood cells, which can deliver oxygen to all body parts, and white blood cells, which help the body fight against bacteria and viruses.
Hematopoietic stem cells (HSCs) live in the bone marrow and serve as the "parent" cells for blood cell formation. They have two key abilities that allow them to perform this role. The first is long-term self-renewal, meaning HSCs can keep making more stem cells like themselves throughout a person's life. The second is multilineage differentiation potential, where HSCs act like seeds that can grow into various "plants," which are the different types of blood cells... (More) - Hematopoiesis is an important biological process that keeps you alive and healthy. This process produces different blood cells, such as red blood cells, which can deliver oxygen to all body parts, and white blood cells, which help the body fight against bacteria and viruses.
Hematopoietic stem cells (HSCs) live in the bone marrow and serve as the "parent" cells for blood cell formation. They have two key abilities that allow them to perform this role. The first is long-term self-renewal, meaning HSCs can keep making more stem cells like themselves throughout a person's life. The second is multilineage differentiation potential, where HSCs act like seeds that can grow into various "plants," which are the different types of blood cells here.
Reactive oxygen species (ROS) are typical byproducts of cellular metabolism and regulators of normal hematopoiesis. Peroxisomes present in all eukaryotic cells and are important for ROS production and catabolism. So, what is the relationship between peroxisomes and HSCs?
This project develops an assay to evaluate the role of peroxisomes in hematopoiesis using advanced gene modification techniques. Genes act as instructions for how cells should work, and by altering these genes, we can change cells' behavior. Manipulating peroxisome gene expression may affect their work patterns and influence ROS levels. These changes could then impact how HSCs function and, in turn, affect normal hematopoiesis.
Using the workflow in this project, genes crucial for peroxisome function can be analyzed to make initial conclusions about their roles in normal hematopoiesis. These findings can then be further tested in animal studies, potentially leading to new strategies to treat blood-related diseases. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9174007
- author
- Wang, Jinyuan LU
- supervisor
-
- David Bryder LU
- Qinyu Zhang LU
- organization
- course
- KIMM01 20241
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Peroxisomes, Hematopoietic Stem Cells, Gene Editing, Lentiviral Vector, CRISPR/dCas9-KRAB System, Flow Cytometry, Mouse Model, immunotechnology
- language
- English
- id
- 9174007
- date added to LUP
- 2024-09-11 08:36:12
- date last changed
- 2024-09-11 08:36:12
@misc{9174007,
abstract = {{Hematopoiesis refers to the formation of mature blood cells, a process driven by hematopoietic stem cells (HSCs) in the bone marrow. These rare cells are characterized by their long-term self-renewal and multilineage differentiation potential, enabling the maintenance and reconstitution of the hematopoietic system. Reactive oxygen species (ROS) are key regulators of normal hematopoiesis, and peroxisomes play a crucial role in ROS production and catabolism, yet their role in hematopoiesis is underexplored. This study establishes an in vitro assay to evaluate the functional roles of peroxisomes in hematopoiesis. The assay employs flow cytometry to precisely identify, analyze, and isolate HSCs. The CRISPRi guide RNA (gRNA) library and a Doxycycline-inducible CRISPR/dCas9-KRAB system enable specific manipulation of peroxisomal gene expression. A polyvinyl alcohol (PVA)-based culture system facilitates the in vitro expansion of HSCs, and gRNA delivery via lentiviral transduction. The functional roles of peroxisomes in HSC maintenance and differentiation are assessed using different in vitro culture systems. Through this workflow, PEX genes, which encode proteins essential for peroxisome function, can be analyzed to draw initial conclusions about their roles in normal hematopoiesis. To further evaluate the stem cell activity of candidate HSCs in vivo, bone marrow transplantation into conditioned (irradiated) wild-type recipients is required. Identifying peroxisomes as a novel metabolic factor in HSC biology could provide a strategy to enhance HSC function through the pharmacological targeting of peroxisome metabolism, offering potential advances to counteract hematological diseases.}},
author = {{Wang, Jinyuan}},
language = {{eng}},
note = {{Student Paper}},
title = {{An In Vitro Assay : To Evaluate the Functional Roles of Peroxisomes in Normal Hematopoiesis}},
year = {{2024}},
}