Haematopoietic Stem Cell Sciences PDF

Challenges of stem cell research How to find the right type of stem cells? How to put the stem cells into the right place? Will the stem cells perform the desired function in the body? Differentiation protocols for many cell types have not been developed.  Chapter 2...

Challenges of stem cell research How to find the right type of stem cells? How to put the stem cells into the right place? Will the stem cells perform the desired function in the body? Differentiation protocols for many cell types have not been developed.  Chapter 2 Properties of Haematopoietic Stem Cells Medical Laboratory Sciences Haematopoietic Stem Cell Sciences Dr. Ali Abdelfattah  Headlines ❑ The hierarchy of haematopoietic cells ❑ HSCs generation ❑ The fate of HSCs ▪ HSCs differentiation ▪ HSCs self-renewal ▪ HSCs mobilization ▪ Apoptosis for HSCs ❑ Cell cycle regulation of HSCs ❑ BM niches ▪ Types of BM niches ▪ Regulation of HSCs by BM niches ▪ Energy source for HSCs ▪ Interplay between HSCs and BM niches ▪ Cancer stem cell niches  Introduction Blood is one of the most highly regenerative tissues, with approximately one trillion cells arising daily in adult human bone marrow (BM) Haematopoietic stem cells (HSCs) reside at the top of the hierarchy of haematopoietic cells and are defined as the cells that have both the self-renewal capacity and the potential to give rise to all hematopoietic cell types (Potency). The frequency of HSCs in BM is about 0.5-2.0% of total nucleated cells ▪ Self-renewal is the ability of HSC to give rise to itself without differentiation. ▪ Potency is the ability of HSCs to differentiate into all functional blood cells (Specialised cells).  The hierarchy of haematopoietic cells HSCs have self- renewal potential and give rise to MPPs that possess limited (MPPs) self-renewal ability but can differentiate into multi-lineage haematopoietic cells CMP: common myeloid progenitors CLP: common lymphoid progenitors MEP: megakaryocyte/ erythrocyte progenitors GMP: granulocyte /macrophage progenitors Potency of haematopoietic cells  HSCs generation SCL, FLI1, LMO2 ,RUNX1, and GATA2 transcription factors are involved in the emergence of HSCs from haemogenic endothelium in process known as endothelial to haematopoietic transition Endothelial to AGM region haematopoietic (aorta-gonad- transition mesonephros) ❖ Transcription factors are proteins involved in the regulation of gene expression by promoting/repressing transcription The fate decision of HSCs  The balance between self-renewal and differentiation Since mature blood cells are predominantly short lived, HSCs continuously provide more differentiated progenitors while properly maintaining the HSC pool size throughout life by precisely balancing self-renewal and differentiation The balance between self-renewal and differentiation is mediated by a complex network of intracellular (transcription factors) and extracellular (BM niches, provided growth factors, cytokines, and signalling pathways) mechanisms that regulate the cell fate.  The balance between self-renewal and differentiation The disruption of the balance between self-renewal and differentiation in the blood production can lead to several haematological diseases. For instance, a defect in the ability of the cells to differentiate can lead to a shortage of blood cells in bone marrow diseases like anaemia, while increased self-renewal can lead to leukaemia. Transcription factors regulate the self-renewal and differentiation capacity of HSCs Several transcription factors regulate the self-renewal potential of HSCs such as GATA2, BMI1, and GFI1, whereas others are involved in differentiation along the major cell lineages. For instance: PU.1 and CEBP commit cells to the myeloid lineage GATA2 and GATA1 commit cells to erythrocytes and megakaryocytes GATA3 and IKAROS commit cells to lymphoid lineage  Epigenetic modifications regulate the self-renewal and differentiation capacity of HSCs Epigenetic is the study of how cells control gene activity without changing the DNA sequence. It refers to external modifications to DNA that turn genes "on" or "off. Epigenetic modifications affect HSCs self-renewal and differentiation through controlling gene expression (up or down regulation) DNA methylation histone modification Demethylation is mediated by the ten- eleven translocation (Tet) proteins  HSCs homing Homing can be defined as the successful attaching of haematopoietic stem cells to the bone marrow before they start to proliferate Homing of HSCs starts from the embryonic period, when HSCs migrate from the fetal liver to seed the bone marrow and repopulate haematopoietic cells of all lineages that are then released into circulation. HSCs recognize, adhere and migrate to the BM through: ▪ CXCR4 (chemokine receptor type 4) ▪ CXCR7 (chemokine receptor type 7) ❖ These proteins are important in guiding HSCs to their final destination  HSCs mobilization Although HSCs mainly reside in the bone marrow throughout adult life, HSCs in substantial numbers leave their bone marrow niches and enter the circulation. Some HSCs in the circulation go through the spleen and liver and perhaps stay within these organs for a while (extramedullary haematopoiesis). A portion of circulating HSCs returns to the bone marrow. Granulocyte colony-stimulating factor (G-CSF) mobilizes HSCs from the marrow into circulation. G-CSF is essential for HSCs mobilization  HSCs self-renewal, differentiation, or death HSCs can undergo other cellular fates such as apoptosis (programmed cell death), and senescence, the latter two resulting in cell death ▪ Apoptosis is necessary for regulation of numbers of HSCs or elimination of damaged cells ▪ Senescence is a process by which a cell ages and permanently stops dividing Apoptotic proteins Pro-apoptotic: enhance apoptosis ( Bak and Bax proteins) Anti-apoptotic: inhibit apoptosis (BCL2 family) Cell cycle regulation of HSCs  Cell cycle regulation of HSCs The HSC activity within the cell cycle is reflected by the developmental demands of the organism ▪ Fetal HSCs are rapidly divided to support the growth requirements, and around 100% of HSCs are activated within the cycle ▪ Conversely, approximately 80% of adult HSCs are in the quiescent phase (resting phase, also known as G0 phase), and around 20% of HSCs enter the cell cycle to maintain blood homeostasis for a long period in adult life ❖ Quiescence of HSCs is critical to protect the stem cell pool from exhaustion under conditions of various stresses to maintain lifelong production of mature blood cells  Cell cycle regulation of HSCs While mature blood cells are produced at a rate of more than one million cells per second in the adult human, most of the HSCs primarily reside in the G0 phase of the cell cycle under homeostatic conditions ▪ Understanding the mechanism of quiescence, self-renewal and differentiation of HSC has been a central issue HSCs undergo either self- renewal or differentiation through cell division Cell cycle regulation of HSCs ❑ Symmetric division ▪ When two daughter cells are stem cells, this division is termed symmetric self-renewal. ▪ When two daughter cells are progenitor cells, this division is termed symmetric differentiation. ❑ Asymmetric division When one daughter cell is a stem cell and the other is a progenitor cell, this division is termed asymmetric  Cell cycle regulation of HSCs HSC HSCs Expansion HSC HSC HSCs Maintenance HPC HPC HSCs Differentiation HSC HPC Quiescence (G0 Phase) The cell cycle phases Interphase G1 phase: grow phase 1 S phase: DNA synthesis phase G2 phase: grow phase 2 Mitosis phase: the cell division phase  Cell cycle regulation of HSCs CDK4/6 CDK2 CDK2 CDK1 CDK1 Cyclin D Cyclin E Cyclin A Cyclin A Cyclin B Self-renewal Maintenance Differentiation HSC Quiescence Cyclin-dependent protein kinases (CDKs), a cell cycle regulatory proteins, encourage HSCs to enter the cell cycle phases through binding to their cyclin partners at each stage  Cell cycle regulation of HSCs To enter the G1-phase, CDK4 and CDK6 interact with cyclin D to form a cyclin-D_CDK4/6 complex to initiate the phosphorylation of retinoblastoma proteins that subsequently activate E2f and allow HSCs to enter the G1-phase ❑ Retinoblastoma (Rb) is family of transcriptional proteins that repress HSCs from entering the cell cycle by suppressing of E2f transcription factor  Cell cycle regulation of HSCs p16 ❑ Cyclin-dependent kinase inhibitors (CKIs) are essential to maintain HSCs in a p18 p19 p21 quiescent state by hindering the activity of p15 p57 p27 CDKs. CKIs are divided into two main categories: ▪ The Inhibitor of CDK4/6 (INK4) family CDK4/6 CDK2 (p15, p16, p18 and p19) that represses the activity of cyclin-D_CDK4/6 complexes Cyclin Cyclin E D ▪ The CDK inhibitory protein/kinase inhibitor protein (CIP/KIP) family (p21, p27, and p57) that suppresses the cyclin- E_CDK2 complex  Cell cycle regulation of HSCs In addition to the cell cycle regulatory proteins, there are transcription factors, growth factors, and signalling pathways, that influence the HSCs fate to maintain the adequate level of blood homeostasis  Proto-oncogenes vs Tumour suppressors Cell cycle is tightly regulated by proto-oncogenes and tumour suppressors Proto-oncogenes are cell cycle positive regulators that enhance cellular proliferation Tumour suppressors are negative regulators that inhibit cellular proliferation  Proto-oncogenes Proto-oncogenes are normal cellular genes that regulate cell growth and differentiation. Proto-oncogene is positive cell cycle regulators, such as Ras and Raf proteins The normal, not-yet-mutated forms are called proto- oncogenes, while the overactive (cancer-promoting) forms of these genes are called oncogenes. So, proto-oncogene can turn into an oncogene if it mutates in a way that increases its activity. Oncogenes drive the cell cycle forward, allowing cells to quickly proceed from one cell cycle stage to the next. This highly regulated process becomes dysregulated leading to cellular transformation  Proto-oncogenes Normally, proto-oncogenes drive cell cycle progression only when growth factors are available. If one of the proteins becomes overactive due to mutation, however, it may transmit signals even when no growth factor is around.  Tumour suppressors Tumour suppressors are proteins that stop cell cycle progression. Tumour suppressors are negative regulators of the cell cycle. Tumour suppressors prevent the formation of cancerous tumours when they are working correctly, and tumours may form when they mutate so they no longer work. One of the most important tumour suppressors is tumour protein p53, which plays a key role in the cellular response to DNA damage. p53 acts primarily at G1 checkpoint controlling G1/S transition (it activates p21), where it blocks cell cycle progression in response to damaged DNA and other unfavourable conditions  Tumour suppressors When tumour suppressor genes don't work properly, cells can grow out of control, which can lead to the development of cancer BM niches  Introduction Adults haematopoietic stem cells (HSCs) reside in the bone marrow (BM) within a specialized micro-environment called HSC niches (BM niches) HSCs in the BM niche are regulated directly or indirectly by many different types of stromal cells such as mesenchymal stromal cells (MSCs), endothelial cells, osteoblast cells, fibroblasts, mature blood cells, CXCL12-abundant reticular cells (CAR cells), and others. Together, these cells forming the extracellular matrix (ECM). ▪ These cells produce substances like glycoprotein, collagen, cytokines, and growth factors that work together to help control HSC quiescence, self-renewal, and differentiation  Types of BM niches Several studies have suggested that HSCs are localised to, at least, two distinct anatomic regions within the BM: An endosteal osteoblastic niche ▪ Adjacent to osteoblast cells ▪ Play a fundamental role in the HSC maintenance (Quiescence and self-renewal) A perivascular endothelial niche ▪ Close to blood vessels ▪ Play a fundamental role in the HSC differentiation  Regulation of HSCs by BM niches The fine regulation of HSCs by the BM microenvironment promotes maintenance of HSC quiescence, which is critical for preservation of their self-renewal potential, HSC self-renewal, and HSCs differentiation HSC niches produce growth factors and adhesion molecules, that encourage HSCs maintenance such as: N-cadherin and osteopontin Angiopoietin-1 (Ang1) Thrombopoietin (TPO) Stem cell factor (SCF) Stromal-derived factor-1 (SDF1, also known as C-X-C chemokine- 12 (CXCL12))  Regulation of HSCs by BM niches ❑ Angiopoietin-1 (Ang1) Ang1 is produced by osteoblastic niche. Ang1 acts on Tie2 receptors expressed by HSCs. Tie2/Ang1 interaction activates the expression of adhesion molecules, such as N-cadherin, & Osteopontin expressed by osteoblasts. This enhanced adhesion between the niche cell and the stem cell contributes to the maintenance of the quiescence of HSCs by maintaining the adhesion of HSCs to the bone ❑ Thrombopoietin (TPO) TPO binds to the myeloproliferative leukaemia protein (c-MPL) receptor, which is expressed by HSCs. TPO/c-MPL interaction is essential for HSCs self-renewal  Regulation of HSCs by BM niches ❑ Stem cell factor (SCF) SCF acts on c-KIT receptor that is expressed by HSCs. SCF confers HSCs self-renewal ❑ Stromal-derived factor-1 (SDF1, also known as CXCL12)) SDF1 interreacts with HSCs through CXC-chemokine-4 receptor (CXCR4). SDF-1/CXCR4 signalling enhances the HSC quiescence ❑ CXCL12 abundant reticular (CAR) cells HSCs are attached to CAR cells via CXCR4 in both endosteal osteoblastic and perivascular endothelial niches. CAR cells secrete a large amount of CXCL12. CAR cells are the prominent component in BM niches and play an essential role in the HSCs quiescence. ▪ CAR cells may play a critical role in mediating migration of HSCs between the osteoblastic and vascular niches. It has been proposed that reduction of CXCL12 expression is one of the mechanisms leading to HSC mobilization  Energy source for HSCs The majority of adult HSCs reside in the BM niches with a hypoxic atmosphere (a low-oxygen environment), in which quiescent HSCs (low cycling HSCs) localise at the highest area of a hypoxic gradient. Thus, they depend on glycolysis (anaerobic respiration), which occurs in the cellular cytoplasm , as the main energy source for quiescent HSCs On the other hand, cycling HSCs settle down at the lowest area of a hypoxic gradient in BM niches. Since the differentiation of HSCs requires a high level of energy, mitochondrial oxidative phosphorylation (aerobic respiration, Krebs-cycle) mediates the ATP generation in HSCs to meet their demands of energy  Energy source for HSCs Reactive oxygen species (ROS) are a harmful product that is produced intracellularly through aerobic metabolism in the mitochondria. ROS are tightly regulated by antioxidant enzymes under normal physiological conditions Oxidative stress is defined as a disruption in the balance between the production of reactive oxygen species (ROS) and antioxidant enzymes. Oxidative stress results in excess production of ROS relative to antioxidant defence and can affect HSCs proliferation, differentiation, aging, and death  Energy source for HSCs Adult HSCs reside in hypoxic niches, are quiescent, have a low metabolic rate, and therefore, generate low levels of reactive oxygen species (ROS) Cancer initiation and progression are associated with oxidative stress by enhancing DNA mutations or increasing DNA damage through increasing the cellular proliferation  Energy source for HSCs Quiescent and self-renewing HSCs maintain low ROS level and reside in hypoxic environment. Mild increase of ROS in HSCs causes lineage differentiation; however, excessive ROS cause stem cell senescence or aging and cell death.  Interplay between HSCs and BM niches HSCs migrate between the osteoblastic and vascular niches in both normal and stress situations. One of the major differences between osteoblastic niche and vascular niche microenvironments is the oxygen level. ▪ A higher oxygen level is expected in vascular niche ▪ A lower oxygen level is expected in osteoblastic niche The dynamic model of the haematopoiesis suggests that: The osteoblastic niche might serve as a reservoir for HSC storage, whereas the vascular niche provides an environment for HSC proliferation and differentiation to produce progenitors and mature blood cells  Cancer stem cell niche Several studies have suggested that there is a functional microenvironment that supports cancer stem cells. Cancer stem cells are derived through intrinsic mutation that leads to become highly proliferative. This highly proliferative state itself alters the signalling balance between niche and the stem cell. Stem cell regulatory systems in the niche for cancer lean more toward the proliferative side than those for normal stem cells. In normal stem cell and its niche, the signalling toward stem cell quiescence and self-renewal is dominant In cancer stem cell and its niche, the proliferative signalling is more dominant