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Stem cells in Hematology; from their Definition and Regulation to their Sources and Use… M.C. le Bousse-Kerdilès Inserm U972, Villejuif - France Hematopoietic Stem Cell Transplantation Training Course ESH-EBMT (Vienna, May 8-10, 2014) Stem Cell Definition and Regulation Canonical definition (From © 2009. Teisha Rowland ) Stem cells are defined by two main properties: (1) Self-renewal (create stem cells “indefinitely”) (2) Differentiation into a diversity of specialized mature cell types Definition Regulation Source and Use Several types of stem cells… Embryonic SC Totipotent (Generate embryonic and extra-embryonic cell types) Ex: Fertilized egg Endoderm Pluripotent Mesdoderm Ectoderm (Generate cells of the three germ layers) Ex: ES cell lines Adult SC Multipotent (Generate several cell types) Ex: Hematopoietic SC Unipotent (Generate only one cell type/tissue) Ex: Keratinocytes Definition Regulation Source and Use Lung Pancreas Muscle Blood Skin Neuron (Modified from Institute of Cell Therapy Website; Ukraine) Stem cell hierarchy during lifespan Loss of pluripotency and proliferative capacity Morula Blastocyst Fetus Embryonic SC (Toti/Pluripotent) Neonate Fetal/Adult SC (Multi/Unipotent) Definition Regulation Source and Use Child Adult Aged Precursors Progenitors 1% Definition 99% Regulation Source and Use Bone marrow (Quiescent) << 0.01% Blood Stem cells CSH/CD34+ Differentiated cells Differentiation Proliferation Hematopoietic stem cells; the archetype of multipotent stem cells Stemness; a hidden property! Hoechst SP Functional markers Rhodamine KI67 Iodure de propidium ALDH NOD/SCID Téloméres Human CSH/PH Tie-2 CD34 Gata 2 CD133 Phenotypic markers Definition Runx1 CD90 Regulation Cell cycle markers Pyronine Y CD38 Notch CD117 Source and Use Oct-4 Molecular markers Regulation of hematopoiesis through extrinsic and intrinsic processes Receptors Cytokines Chemokines HSC/CD34+ Transcription factors Signaling pathway Endothelial cells Definition Regulation ECM GAGs ECM receptors Adhesion molecules Stromal cells Source and Use Transcription factors regulate gene expression by binding to specific sites on DNA Transcription start site Binding site (proximal or distal) Mechanism dependent on the 3D structure of the chromatin that is regulated by epigenetic modifications Definition Regulation Source and Use (From Duke University website) Molecular mechanisms mediating epigenetic regulation 1) DNA methylation that establishes a compact/silent chromatin state 2) Histone post-translational modifications - acetylation - methylation, - phosphorylation - ubiquitination/sumoylation TF 3) Small RNAs that can regulate the histone code Epigenetic modifications are reversible and highly responsive to micro-environmental signals Definition Regulation Source and Use (From Alan Horsager, 2014) The Bone Marrow, a specific environment in adult Endosteum Vascular sinus Definition Regulation Source and Use Hematopoietic stem cell niches Endosteal niche Mesenchymal niche - Maintain HSC quiescence - Trigger HSC activation and proliferation - Participate in HSC mobilisation out of the BM Vascular niche Definition Regulation - Cell-Cell interactions - Adhesion molecules - Cytokines/Chemokines - Proteases - Extra-cellular matrix components - Environmental factors (Ca++, O2/perfusion..) Source and Use (Paul S Frenette et al, Annu, Rev, Immunol, 2013; Lapidot, ASH 2012;) When niches become creasy…. Homeostasy Vacancy/Aplasia Proliferation/Leukemia Definition Regulation Source and Use (Scadden D.T. ; Nature 441, 1075-1079, 2006 Walkley et al; Cell 2007; Kim et al, Blood, 2008; Lataillade et al; Blood 2009; Raaijmakers et al, Nature 2010) Stem Cell Sources and Use for Cell Therapie and Regenerative Medicine Embryonic vs. Adult stem cells Advantages and disadvantages for cell therapies Embryonic vs. Adults Stem cells Advantages and disadvantages for cell therapies Totipotent Embryonic SC Pluripotent (blastocyst) Multipotent (embryonic ES cells tissues) - Transformed cell products (ESC: in vitro derived from blastocysts) - Destroyed source - Pluripotent Neonate Adult SC Adult Aged - Untransformed cell products (HSC, MSC, EPC…) - Undestroyed source - Multipotent Embryonic vs. Adults Stem cells Advantages and disadvantages for cell therapies Totipotent Embryonic SC Pluripotent (ES cells) Multipotent (embryonic tissues) - Transformed cell products (ES cells; in vitro artifacts) - Destroyed source - Pluripotent Neonate Adult SC Adult Aged - Untransformed cell products (HSC, MSC, EPC…) - Undestroyed source - Multipotent Bone marrow: major source of adult stem cells Pluripotent somatic stem cells ? HSC Hematopoietic stem cells (1/105) ? Neuronal cells EPC Endothelial stem cells (≤1/106) Mesoderm Endoderm Ectoderm Stromal Cells MSC ? Hepatocytes Osteocytes Chondrocytes Mesenchymal stromal cells (1-5/106) Definition Regulation Sources & Use Adipocytes Other sources of adult stem cells… Adipose tissue Mobilized peripheral blood Liposuction Sources of: - HSCs - MSCs - EPCs… Sources of SC capable to differentiate into cells from meso-, ecto-, and endo-dermal lineages In mouse model (Baer et al., Stem Cells Int., 2012; Pitchford et al., Cell Stem Cell 2009) Stem cells from perinatal tissues Umbilical Cord Blood Sources of - MSCs - EPCs - HSCs - ES like (VSELs…) Definition Amniotic membranes Regulation Sources & Use Cell therapies and Regenerative medicine Differentiated cells Immediate but short term repopulating capability (Ex: RBC, Pl) “ Specialized” stem cells Long term repopulating capability but restricted properties (Ex: HSC, EPC) “Generalist” stem cells Multipotent properties Action on environment (Ex: MSC) Definition Regulation Sources & Use Mesenchymal Stroma Cell (MSC); a drug cell for regenerative medicine Present in numerous organs/tissues Multipotential properties Action on environment - Immuno-modulatory - Anti-inflammatory - Anti-fibrotic - Anti-septic Currently used in pre-clinical application and in some clinical trials: - GVHD - HLA-mismatched HSC graft - Osteoporosis, Bone defect - Skin burns - Vascular regeneration (in combination with EPCs) Definition Regulation Sources & Use (Arien-Zakay et al; Best Practice & Research Clinical Hematology;2010) Endothelial Progenitor Cell (EPC) origin and use for cell therapy Bone Marrow Peripheral Blood Neoangiogenesis Umbilical Cord Blood Damaged ischemic tissue Definition Regulation Sources & Use HSC amplification without loosing their multipotentiality… the quest for Holy Grail! Increasing HSC amplification • • Awakening dormant HSC (cytokine cocktails, inhibitors of Cdki …) Inhibiting their differentiation (activation of Notch signaling/engineered Notch • Using regulators of self-renewal/differentiation balance • Providing a niche by coculture with MSC (transplant of 2 CB units, one of which ligands, of Wnt pathway, BMPs…) hydrocarbon receptor antagonist, copper Chelator, PGE2, HOXB4, cytokines …) (Aryl ex vivo expended in MSC coculture) Increasing HSC homing • • • Definition Intra-bone injection Agents modulating chemokine/receptor axis (CXCR4…) Agents inhibiting the peptidase CD26 … Regulation Sources & Use (Dahlberg et al., 2011; Körbling and Freireich.,2011; De Lima et al., 2012) Embryonic vs. Adults Stem cells Advantages and disadvantages for cell therapies Totipotent Embryonic SC Pluripotent (ES cells) Multipotent (embryonic tissues) Neonate Adult SC Adult Aged Use of embryonic stem cells in regenerative medicine - As pluripotent, it is possible to obtain all mature cell types from ES cell differentiation - Once differentiated, they can be used to treat degenerative diseases of many tissues and organs - Transformed cell products - Destroyed source - Ethical and religious concerns Back in time… with induced Pluripotent Stem Cells (iPSC) ! Totipotent Embryonic SC Pluripotent (ES cells) Multipotent (embryonic tissues) iPSC Yamanaka & Gurdon Neonate Adult SC Adult Aged - Characteristics close to those of ES cells - Re-programming adult somatic cells to pluripotent stage (Thomson et al; Science 2007; Yamanaka et al; Cell 2007) Reprogramming adult somatic cells to pluripotent state 1% (http://www.eurostemcell.org) Reprogramming adult somatic cells to a pluripotent state by forced expression of pluripotency genes or … by different stresses such as physical squeezing or submersion in weak acidic solution (STAP cells “Stimulus-Triggered Acquisition of Pluripotency” (Obokata et al, Nature; 2014)) A place for ES and iPS cells in hematology? Experimental research and clinical applications Drug screening In vitro models of human development In vitro models of genetic diseases Cell-based regenerative medicine Definition Regulation Sources and Use (Schlaeger and Guan; 2009) From human ES/iPS cells to blood cells and…from blood cells to human iPS! hESC Specialization ? hiPSC Reprogrammation CD34+ (Oct-4, Sox2 and Klf4, c-Myc, or Nanog, Lin28, other stimuli ?….) Definition Regulation Sources & Use (mESC: Evans & Kaufman, Nature 1981 miPSC: Yamanaka, Cell 2006 and Thomson, Cell 2007) First clinical trial: Blood made from stem cells will be tested in 2016-17 in UK! 2010: First generation of red blood cells from human ES/iPS (Lapillonne et al., 2010)…. Difficulties in enucleation (J. O’Callaghan, Scotland; MailOnline 14 April 2014) (Scottish National Blood Transfusion Service) 2014: Biochemical environment to produce enucleated RBC 2016-2017: First in-man trial in that will probably use ES cells, but will move on to iPS cells because it’s easier to choose the blood group (O- : “Universal donor” )… Towards « artificial » Universal Blood? 1667 From lamb… to human ES or iPS cells! JB Denys, physician of King Louis XIV of France Three and a half centuries later… 2030...?