Stem Cell Biology and Haemopoietic Differentiation PDF

Stem Cell Biology and Haemopoietic Differentiation Instructors : Professor Amein Al-Ali email: [email protected] Department of Clinical Biochemistry Monday, December 11, 2023 Essential Concepts • Stem Cells • Haemopoietic Niche • Types of Stem Cells • Transdifferentiation • Regulation of Haemo...

Stem Cell Biology and Haemopoietic Differentiation Instructors : Professor Amein Al-Ali email: [email protected] Department of Clinical Biochemistry Monday, December 11, 2023 Essential Concepts • Stem Cells • Haemopoietic Niche • Types of Stem Cells • Transdifferentiation • Regulation of Haemopoietic Differentiation • Growth Factors • Transcription factors Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 2 Learning objectives Can we use our portfolio of antibodies to cell- surface markers to identify signatures of stem cells and their derivatives outside hematopoiesis? Monday, December 11, 2023 Presentation title Presentation title 28th 3 End products of Hematopoiesis 6 T.ie n differintiatemoooh.d Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title Final result Final purpose of tree Cannot multiply End Product Reason for haemopoiesis No proliferation (mostly) Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 5 5 Stem Cell Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 6 Components of Haemopoiesis • Seeds = Stem cells • Soil = BM ‘Niche’ • Fertilizer = Growth + H2O factors cell seeds stem drifter Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title In faro 7 Stem Cell Niche  Composed of microenvironmental cells that (perivascular-Around or surrounding a blood vessel) the function of which are: – Nurture stem cells – Protect from apoptosis, – Prevent excessive differentiation (loss of reserve) – Prevent excessive proliferation (cancer risk) • Mesenchymal stromal cells, nerve cells, osteoclast, osteoblasts & immune cells are main haemopoietic ‘niche’ cells Monday, December 11, 2023 Medicine 1 Lecture 3 P100 - P101 Presentation title 8 •Mesenchymal stromal cells, nerve cells, osteoclast, osteoblasts & immune cells are main haemopoietic ‘niche’ cells Factors SCF- Stem cell factor required for maintenance CXCL12 - required for maintenance & retention TGFTransforming growth factor beta Thrombopoietin- required for maintenance and platelet production TGF-Transforming growth factor beta SCF- Stem cell factor Other factors- Angiogenin, fibroblast growth factor (FGF), interleukin 6, MSC: mesenchymal stem cells Lepr-expressing perivascular cells maintain haematopoietic stem cells Monday, December 11, 2023 Presentation title Stem cells within their niche in the bone Factors marrow SCF- Stem cell factor required for maintenance CXCL12 - required for maintenance & retention Thrombopoietin- required for maintenance and platelet production TGF-Transforming growth factor beta Other factors- Angiogenin, fibroblast growth factor (FGF), interleukin 6, Stromal cells LEPR- leptin receptor cells express CXCL12 & LEPR CAR - CXC-chemokine ligand 12 (CXCL12)-abundant reticular cells –express CXCL12 9 Monday, December 11, 2023 Presentation title Properties of Stem Cells • Self renewal – the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. • Potency – the capacity to differentiate into specialized cell types. Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title Potency • Totipotent stem cells - can form any cell of the embryo (embryonic stem cells-Morula) • Pluripotent – differentiate into cells derived from the three germ cell layers (HSC) • Multipotent – can produce cells of a closely related family of cells CLP-common lymphoid progenitor, T & B cells, NK cells CMP-common myeloid progenitor, erythrocytes, platelets, monocytes, base, neutron and eosinophil) • Unipotent –only produce one cell type, but have the property of self renewal which distinguishes them from the non stem cells Cells with the greatest potency can generate more cell types than those with lower potency. non stem cells Monday, December 11, 2023 Medicine 1 Lecture 3 P100 - P101 Presentation title 11 Monday, December 11, 2023 Presentation title 28th 13 The Major Types of Stem Cells A. Embryonic Stem Cells • From blastocysts left over from In-Vito Fertilization in the laboratory • From aborted foetuses B. Adult Stem Cells • Stem cells have been found in the blood, bone marrow, liver, kidney, cornea, dental pulp, umbilical cord, brain, skin, muscle, salivary gland … Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title Advantages and Disadvantages to Embryonic and Adult Stem Cells. Embryonic S.C. Adult S.C. “Pluripotent” (can become any cell) “Multipotent” (can become many but not any) Less Stable. Capacity for self-renewal is limited. Difficult to isolate in adult tissue (except haemopoietic) Host rejection minimized Stable. Can undergo many cell divisions. Easy to obtain but blastocyst is destroyed. Possibility of rejection?? Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 • Blood is collected from umbilical cord immediately after delivery about 100150cc • The number of cells in 1 ml is 40,000 • They are stored in blood banks at -19C in a state of suspended animation and restart their activity on thawing CD 34, 1-5% of cord blood and 0,001-0,01% of peripheral blood cells Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 15 Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 15 Shinya Yamanaka Medicine 2012 Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 16 Genetic manipulation in animals Transgenic v/s knock-out Animals Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 17 Transgenic • A transgenic animal is one that carries a foreign gene (DNA) that has been deliberately inserted into its genome artificially. • DNA introduced into Embryonic Stem Cells artificially • DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum – Random (approx.. 10% disrupt an endogenous gene important for normal development) - Multiple copies Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 17 Monday, December 11, 2023 Transgenic Mice Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 19 Transgenic Mice Brain has a set of receptors known as NMDA which help with memory formation and learning & controlled by NR2B when young and NR2A when old. Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 19 Transgenic Mice •Transgenic mice are useful for: 1. characterize the ability of a promoter to direct tissue-specific gene expression 1. – e.g. a promoter can be attached to a reporter gene such as LacZ or GFP (Jellyfish- green fluorescent protein ) 2. examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 19 Transgenic sheep generation • Eggs are harvested from 0.5 dpc (superovulated or natural matings) (days post coitum) • The test DNA is injected into the male pronucleus • The eggs can be transferred the same day or the next (2-cell) into pseudopregnant female oviducts – A sheep in estrus is mated with a vasectomized male Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title Pronuclear Injection Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 25 Study effects of Genes/proteins Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 20 Study ‘reporters’ Green Fluorescent Protein Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 22 Study activity of Promoters of genes Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 21 Ectopic eye development by transgene expression Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 23 knock-out animals A knockout mouse is a genetically modified mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. • DNA is introduced first into embryonic stem (ES) cells. ES cells that have undergone homologous recombination are identified and injected into a 4 day old mouse embryo - a blastocyst – targeted insertion Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 18 Integration is by homologous recombination Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Knock-out mice • Used to study the physiological role of a protein of interest in growth and development by deleting it (“knocking- out”) in embryos • ‘Unconditional KO’: Gene deleted in all tissues • ‘Targeted KO’: gene deleted only in specific tissues (eg only heart, or brain, rather than all organs) and at different time- points (eg at birth, only after a certain age) Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 26 Cloning by manipulation of Embryonic Stem Cells • Isolate ES cells from ‘inner cell mass’ of embryos • Culture cells in vitro (these are available as cell lines for mice strains) • Introduce new genes or alter existing genes in the cultures ES cells (homologous recombination) • Select cells which have gene alteration – Positive selection (antibiotic) – Negative selection (gancyclovir to kill-off ‘Tk) • Transfer them back into other embryos Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 28 Chimeric Mice Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title Dolly http://repairstemcell.wordpress.com/2009/02/07/the-associated-press-stem-cell-cloning-expert-jerry-yang-dead-at-49/ Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 30 Reproductive Cloning: Somatic Cell Nuclear Transfer (SCNT) Pre-implantation Genetic Diagnosis Screening for gene expression and imprinting defects Enucleation Infertile Male Clhild Post-implantation Genetic Diagnosis screening for gene integrity and imprinting defects Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Ethical issues • Are we trying to play GOD? • Is embryo a person? • Will stem cell research encourage embryo destruction and abortions? Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 http://www.biotechnology.gov.au/assets/documents/bainte rnet/StemCells2006820060502132032.pdf Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 33 Graf T, Blood, 1 May 2002, Vol. 99, No. 9, pp. 3089-31 Summary of hematopoietic-to-nonhematopoietic cell conversions The scheme summarizes experiments in which bone marrow cells were transplanted into mice, which were then analysed several months later hematopoietic and nonhaemopoietic donor type cells. Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 Presentation title 34 Stem cell plasticity ASC; Adipose-derived stromal/stem cells NSC; Neural stem cells Transdetermination - a switch in lineage commitment in a stem or progenitor cell Immunological Rev 187:22-39 Transdetermination Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 35 Medicine 1 Lecture 3 P100 - P101 Monday, December 11, 2023 35