10 0 101 102 103 104 SCA1-Height R Hematopoietic stem Totipotent Stem Cell Zygote Embryonic und adult tissue stem Pluripotent Stem Cell Embryonic Stem (ES) Cells T-CELL LYMPHOID STEM CELL B-CELL PLASMA CELL Multipotent Tissue Stem Cells BFU-E ERYTHROCYTE Aleksandra Wodnar-Filipowicz Department of Biomedicine University Hospital Basel Switzerland ESH-EBMT Targu Muresh 2010 1 Neural Muscle Liver Epidermis Retina Hematopoietic Cancer Stem Cells MYELOID STEM CELL CFU-GM CFU-Eo CFU-Baso CFU-Meg NEUTROPHIL MONOCYTE MACROPHAGE EOSINOPHIL BASOPHIL PLATELETS 2 MEGAKARYOCYTE Hematopoietic stem Presentation outline: 1. Bone marrow hematopoietic stem 2. Bone marrow stem cell 3. Leukemia stem 4. Embryonic/pluripotent stem! Therapeutic concepts c-kit c-kit Mouse Phenotype Lin - LSK sca1 + 0.007% ckit + of BM tie2 + flk2 - CXCR4 + ABCG2 + SLAM-Rs: CD150 + CD244 - CD48 - TLR4 + BL6 % LSK : 0.007% sca-1 Human Phenotype Lin - CD34 + CD38 - ckit + flk2 - CD133 + CXCR4 + ABCG2 + Side population (SP) (ABCG2 + transporter) Hoechst blue Hoechst Blue 0.2% SP Hoechst Far Red Hoechst far red CD34 + CD38-0.1% of BM 3 4 Human hematopoietic stem In vitro CFU assays Function In vivo transplantation: xenografts in immunologically permissive mouse strains (e.g...nod/scid; NOD/SCID-!c-/-; Rag2-/-!c-/-) SRC human CD34 + (i.v. or i.f.) Bone marrow stem cell s CFSE/ DAPI/ OPN bone BM os endosteum Wilson et al. Genes & Dev 2004 c-kit + LRC + marrow BMT BMT 1º 2º 3º endosteum Clinical transplantation 5 6
Stem Cells - Definition: Cellular components of stem cell s in the adult bone marrow Cells capable to: self-renew i.e. produce undifferentiated differentiate and give rise to specialized Stem Cell Niche - Definition: Supportive bone marrow microenvironment structures that are essential for the long-term maintenance of a stable pool The is anatomically and functionally defined to have: endosteal compartment: proximity to trabecular bone perivascular compartment: proximity to sinusoidal Trabecular bone Endosteal Sinusoid endothelial (SEC) Precursor Osteoblasts - osteogenic CAR - (CXCL12 abundant vascular endothelium 7 lining the inner surface of the bone reticular) close to 8 the bone surface and endothelium CAR Osteoblasts Vascular Stroma fibroblasts Molecular interactions in the bone marrow Extrinsic Intrinsic mechanisms: Sinusoid mechanisms: cytokines endothelial (SEC) signalling molecules chemokines negative regulators (osteopontin) adhesion molecules VEGFR2 & 3 - VEGF cytoplasmic nuclear, incl. transcription factors proteases (MMP-9, cathepsin K) hormones (PTH, PGE2) epigenetic mechanisms DNA/histone modif. sympathetic nerves micrornas Gene expression oxygen status calcium concentration circadian rhytm quiescence self-renewal expansion Ang-1 - tie2 SCF - c-kit Flt3-L - Flt3 TPO - mpl Wnt - Frizzled Jagged/Delta - Notch OSTEOBLAST PTH, PGE2 Cadherins VCAM - VLA ICAM - LFA CXCL12 - CXCR4 HA - CD44 nerves cell-cell adhesion migration 9 Functional interactions in the bone marrow Hematopoietic homeostasis Migration Proliferation Differentiation Self-renewal Vascular Endosteal Engraftment Regeneration 5-FU IR Stress response Myelosuppressive injury damages SECs but not osteoblasts Administration of angiogenic factors may promote hematopoietic reconstitution after myeloablation through stabilization of vessels (VEGFR2 + VEGFR3 + sca110 - SEC) Fetal stem cell s Human placenta : reside in proximity to fetal blood vessels (pericytes and endothelial ) Robin et al. Cell 5:385-395 (2009) Mouse fetal liver : reside in perisinusoidal sites Iwasaki et al. Blood (2010) Stem cell quiescence: to prevent the stem cell exhaustion to protect stem from acquiring mutations leading to malignant transformation Developmental switch: fetal vs adult Bowie et al. PNAS 104:5878-5882 (2007) birth 3w 4w E14.5 adult Developmental Actively cycling expansion High self-renewal High GM output Quiescent Lower self-renewal Reduced GM output Homeostatic maintenance 11 Migration from active (vascular) s towards BM dormant (endosteal) s 12
reversibly switch from dormancy to self-renewal during homeostasis and repair Wilson et al. Cell 135:1118 (2008) Lin-Sca1 + CD117 + CD135 - CD34 - CD150 + CD48 - Homeostasis ± 15% ± 85% c-kit + LRC + marrow endosteum Dormant : - reduced metabolism & ribosomal biogenesis - reduced DNA replication (to protect from errors) - housed in dormant s (endosteal) as single - represent a reserve pool which can be used upon BM injury Dormant (d) LRC- activity high CD34 mrna low Injury Activated (a) Non-LRC- activity low CD34 mrna high Activated : - designated to proliferate & differentiate - present in vascular s where more oxygen is available to support the increased metabolism and cell-cycle activity < 1000 / mouse 5 divisions / life (mouse) every 145 days Injury: G-CSF, 5-FU 13 14 IFN" activates dormant hematopoietic stem in vivo Essers et al. Nature 458:904-908 (2009) IFN" Leukemia Stem Cells Precursor (d) (a) Endosteal IFN" promotes the cell-cycle entry of (dormant and activated) resulting in proliferation, expansion, differentiation 15 16 Cancer stem Normal stem and cancer share the ability to self-renew and many signaling pathways involved in the regulaton of normal stem cell development are mutated or epigenetically activated in cancer. Cancer Initiating Cell Models Cancer stem cell model Leukemia stem : Transformed hematopoietic stem or commited progenitor that have amplified or acquired the capacity for self-renewal, albeit in a poorly regulated fashion. Prog. 17 Wang and Dick, 2005 18
Leukemia stem : 1 st identified cancer stem Induction of cell cycle entry eliminates human leukemia stem in a mouse model of AML Saito et al. Nature Biotech 28:275-280 (2010) Phenotype Function CD34 + CD38 - CD34 + CD38 + CD33+ CD44+ CD47+ CD71+ CD96+ CD117- CD123+ CD133+ CLL-1+ Human AML peripheral blood Serial assays: AML SL-IC CD34 + CD38 - NOD/SCID BM BM 1 o 2 o 3 o 1 o CFU 2 o CFU 3 o CFU 19 Combined G-CSF and AraC treatment effectively clears endosteal of 20 Leukemic stem resistant to conventional treatment lead to relapse and fatal outcome Current (novel) therapeutic studies Conventional treatment Leukemic cell growth Relapse Induction of cycling: IFN", G-CSF Targeting homing: CXCR4 antagonists Elimination of Degeneration Remission Interfering with adherence: Targeting surface markers: anti-cd44 mab anti-il3 r (CD123) mab Terminal differentiation Remission Preventing immune evasion: anti-cd47mab 1. Activation of dormant IFN"/G-CSF 2. Chemotherapy Targeted therapy 21 22 CD47 is an adverse prognostic factor and therapeutic antibody target on human AML stem Jaiswal et al. Cell 138:276-285 (2009) Majet et al. Cell 138:286-299 (2009) SIRP" CD47 = No phagocytosis SIRP" CD47 Low levels of CD47 on avoid macrophages upon mobilization w. G-CSF High levels of CD47 on enhance survival due to decreased clearance..by macrophages General goal: creation of tumor non-permissive microenvironment BM is a dynamic microenvironment with high concentration of growth factors necessary for hematopoiesis, making it a highly permissive zone for cancer cell homing and survival. This is supported by the fact, that solid tumors very frequently home and metastasize to the BM. Hijacking the Elimination of Differentiation Remission BM stroma or Activation 1. of dormant IFN"/G-CSF 3. 2. Prevent immune evasion Promote Chemotherapy immunosurveillance23 Targeted therapy Seed or soil or both? 24
Generation of ES cell lines from human blastocysts The challenge of embryonic stem cell research: Thomson et al. Science 282:1145 (1998) Kaufman et al. PNAS 98:10716 (2001) Differentiation in vitro into various tissues! ES! ips! Therapeutic outlook Donated IVF blastocyst Inner cell mass immortal cell lines CFU s 25 enucleated Donated donor oocyte oocyte nuclear-transfer from adult cell Therapeutic Cloning (SCNT) Human ES with patient s genetic information = Immunologically-compatible tissue for transplantation 26 Human ES Goal: generation of engraftable hematopoietic Clinical use of ES-derived must address both maintenance and control of pluripotency, and also control of rejection danger. Control of growth and differentiation: Pluripotency may be a double-edged sward, since such have an increased potential for malignant transformation (teratoma formation). Uncontrolled differentiation of hes may lead to undesired progeny, overgrowth, or death of transplanted. Control the rejection: The likelihood of rejection requires strategies to circumvent or control the immune barriers (eg.with tolerogenic ES-derived cell lineages produced in parallel). 27 Human ips (induced pluripotent stem ) from adult dermal fibroblasts Oct3/4 Sox2 c-myc Klf4 fibroblasts Oct4 Sox2 Nanog Lin28 Takahashi et al. Cell 131:1 (2007) Yu et al. Science 318:1917 (2007) Zhou et al. Cell Stem Cell 4:381 (2009) Fusionproteins ± 20 days ips (pips) ES-like Differentiation into of all germ layers: Ectoderm Mesoderm Entoderm " Human ES morphology " Normal karyotype " Telomerase activity " Cell surface markers of ES " Gene expression profile of ES e.g. neural, heart muscle 28 Proof-of-principle papers on the therapeutic potential of ips 1 st report on correction of a disease in mouse : Treatment of sickle cell anemia mouse model with ips generated from autologous skin. Hanna et al. (Jaenisch s lab) Science 318:1920 (2007) Hu sickle cell anemia mouse h#s/h#s 70% PB were derived from ips Diff. to hematopoietic progenitors tail fibroblasts + Oct4/Sox2/klf4/c-Myc viruses ips ips Correction by gene targeting 29 30
1 st report on patient-specific corrected and reprogrammed ips : Disease-corrected haemopoietic progenitors from Fanconi anaemia ips Raya et al. Nature 460:53 (2009) Dermal fibroblasts 6 FA patients 1. Correction of defect lentiviral vectors: FANCA FANCD2 Succesful with: 3 FA patients-derived somatic 12/28 reprogramming attempts CD34 2. Reprogramming Viral vectors: Oct4 Sox2 Klf4 c-myc O nm MMC 1O nm MMC 31 ES SCNT Prospects for stem cell-based therapy of degenerative diseases ips CB- Stem cell choice: adult (multipotent) or embryonic (pluripotent) off-the-shelf from master-stocks (= cell banks) or customized for.........individual patients Stem cell number: increase homing and engraftment in tissue of choice develop drugs that augment the endogenous stem cell pools Stem cell immunogenicity: generate isogenic (genetically equivalent) : ips ES after nuclear transfer ( therapeutic cloning ) parthenogenetic embryos as ES cell source (pes) overcome the immune response: engineer deficient in class I...and II HLA genes/nk ligands or antagonizing immune responses Stem cell gene therapy: ES/iPS terato/cancerogenicity: control insertional mutagenesis 32 NSC Literature - Reviews: Stem - all aspects: Issue of Cell 132; Feb 22 (2008) and s: Kiel & Morrison, Nature 8:290-301 (2008) Jones & Wagers, Nature 9:11-21 (2008) Butler, Kobayashi & Rafii Nature 10:138-146 (2010) Trumpp, Essers & Wilson Nature Revs 10:201-209 (2010) Leukemia stem Chan & Huntly, Seminars in Oncology 35:326 (2008) Leukemic stem Lane, Scadden and Gilliland Blood 114:1150-1157 (2009) Cancer stem - targeting Trumpp & Wiestler, Nature Clinical Oncol 5:337 (2008) Sipkins NEJM 361:1307-1309 (2009) Goff & Jamieson Cell Stem Cell 6:296-297 (2010) 33