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HEMOPOIESIS  Arnold D Rubin, MD Cancer Institute of New Jersey Jan , 2009
HEMOPOIESIS <ul><li>Maintaining the optimal numbers of formed elements in the circulation: RBC for oxygen carrying,WBC for...
How Can You Fulfill these Requirements? Only a mechanism involving stem cells could provide the plasticity and a life long...
CELL DEVELOPMENT STEM STEM STEM STEM STEM Hierarchic Stochastic Hemopoiesis Tumors
STEM CELLS <ul><li>A stem cell must be capable of self renewal and should not be “used up” </li></ul><ul><li>It must be ca...
Definition of a Hemopoietic Stem Cell 1 Repopulate the marrow of a lethally irradiated  animal 2 Have the capacity for sel...
Replenish old or damaged tissue by limited proliferation and maturation Stem cell Differentiation leading to senescence an...
Genetics of Differentiation TF Silent  HOX  DNA  RNA transcribed Translation into proteins to drive differentiation in to ...
Identification and Function of Stem Cells Cell Surface Markers Functional Assays
Concept of Stem Cells Hemopoietic lethal radiation BM cells spleen  colonies mouse Till- McCullogh Preparation CFU-S 8 day...
Maintainance of “Stemness” The length of the telomer determines the ability of the stem cell to keep from differentiating ...
M. Pera, ASH Education,2002
M. Pera, ASH Eduation,2002
M. Pera, ASH Education,2002
Hemopoietic Stem Cells- Surface Characteristics CD34-/lo CD34+ Sca-1+ Sca-1+ Lin- Lin- C-kit+ C-kit+ Low self renewel Mult...
Characteristics of  Stem Cells Properties CD34++ Thy-1+ SSC-low c-kit+ HLADR- CD45RA- CD38- lin- CD13- CD33- in vitro  Ass...
Culture of Clonogenic Progenitors nutrients medium “ gamisch”: CSF’s lineage specific Interleukins semisolid suspension te...
 
NOD-SCID Mouse Inject human stem cells CD38 CD45 Blood for FACS to show human cells  grafted Assay for Human Stem Cells
Ontogeny of Hemopoiesis   <ul><li>Primitive – transient in yolk sac, fetal Hb RBC -7.5 dpc (mouse) </li></ul><ul><li>Migra...
Orkin,S, Cell,2008
Orkin,S, Cell,2008
Other Cells and their Products Interact with Stem Cells
THE JACK SPRAT RIDDLE <ul><li>Marrow cells from the w/w mouse will not engraft a normal mouse </li></ul><ul><li>This mouse...
The Steel Factor-Stem Cell Stromal Interaction Bone Marrow Stroma SLF SLF normal w/w sl/sl c-kit c-kit ( ( c-kit c-kit ( (...
The Big Questions <ul><li>Infused marrow cells know to go directly to the bone marrow </li></ul><ul><li>No matter how many...
The Concept of a Hemopoietic NICHE <ul><li>BONE NICHE – Maintenance of long term quiescience of hemopoietic stem cells in ...
Mutant Mice and  the Bone Niche Runx-2 -/-  mutant lacks osteoblast formation. Consequently no hemopoiesis is found in the...
Orkin,S, Cell,2008
Copyright ©2006 American Society for Clinical Investigation Yin, T. et al. J. Clin. Invest. 2006;116:1195-1201 Hematopoies...
Marrow Stroma HSC BMP Interaction HSC SC SCF  c-kit Jagged1  Notch Ang1  Tie-2 Cadherin  βcatenin VCAM  integrin Osteopent...
Copyright ©2005 American Society of Hematology.  Copyright restrictions may apply. Taichman, R. S. Blood 2005;105:2631-263...
Copyright ©2005 American Society of Hematology.  Copyright restrictions may apply. Taichman, R. S. Blood 2005;105:2631-263...
Copyright ©2005 American Society of Hematology.  Copyright restrictions may apply. Taichman, R. S. Blood 2005;105:2631-263...
Zhang,Nature,2003
Zhang,Nature,2003
The Vascular Niche <ul><li>More support for proliferation and differentiation of HPC’s by sinusoidal endothelial cells </l...
Factors Involved in the Vascular Niche <ul><li>SDF-1 is a homing factor, inducing HSC’s  to transendothelial migration med...
Copyright ©2006 American Society for Clinical Investigation Yin, T. et al. J. Clin. Invest. 2006;116:1195-1201 The osteobl...
Zhu,Oncogene,2002
Transcription Factors These factors play a vital role in both embryogenesis and lineage restricted differentiation  of hem...
Orkin, SH, Cell, 2008
Orkin,SH, Cell, 2008
Blood Cell Development Lympho-myeloid growth depends on a hierarchic array of  clonogenic cells with widely varying potent...
Hierarchy of Myeloid Cell Production S S gemm gemm GM GM GM GM Stromal Cell Macrophage T-cell CD34 +  Sca +  Lin -  Thy-1 ...
Hemopoietic Cytokines Metcalf designed the system to demonstrate the existence and specificity of the various cytokines im...
Culture of Clonogenic Progenitors nutrients medium “ gamisch”: CSF’s lineage specific Interleukins semisolid suspension te...
 
Copyright ©2008 American Society of Hematology.  Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology.  Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology.  Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology.  Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology.  Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
Passegue,PNAS,03
Bruno,Mol & Cell Biol,2004
Stem Cell Transplantation(BMT) BMT can be used to replace a failed marrow or correct certain genetic defects. The adoptive...
ANNUAL NUMBERS OF  BLOOD AND MARROW TRANSPLANTS  WORLDWIDE 1970-2002 NUMBER OF TRANSPLANTS YEAR 1970 1975 1980 1985 1990 1...
stem cells T cells stromal  myeloid progenitors cancer cells other cells Bone Marrow Transplantation BMT is composed of di...
Sources of Stem Cells Source Bone Marrow Stimulated Blood  1/4-1/5  8-9 Cord Blood  slightly  increased  20-46 Fetal Liver...
Nonmyeloid Stem Cells in vitro  expansion of CD34 + , CD45RA low  CD71 low  By IL-3, IL-6 SLF & EPO provides an expansion ...
Stem Cells from Sources Other than Bone Marrow <ul><li>Bood has 10-100 fold fewer stem cells as compared to marrow.  But s...
 
 
Adult Stem Cell Plasticity Hemic Stem cells Mesenchymal Stem Cells Liver Neurons, glial cells Cardiac Fibroblasts Osteobla...
C. M. Verfaillie, ASH Education, 2002
ex vivo Stem Cell Expansion <ul><li>Efficiency of harvest.  At times this might be the only way to obtain sufficient cells...
ex vivo Expansion of Stem Cells <ul><li>Incubate with stroma.  Porcine stroma supports 5 fold expansion in 5 weeks. Maybe ...
Retroviral Vectors Used to Transduce Stem Cells helper DNA gag pol env gag pol env  vector viral RNA proteins helper virus...
 
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  • Slide 1: Estimates of the annual numbers of blood and marrow transplants worldwide extrapolated from data compiled by the National Marrow Donor Program (NMDP), the European Blood and Marrow Transplant Group (EBMT), independent market surveys, U.S. hospital discharge data and data reported to the IBMTR. The past few years has seen a slowing in the growth of both autologous and allogeneic transplants. The drop in autotransplants was due to a decrease in their use for breast cancer. The flattening in growth for allotransplants results from a decreased in their use for chronic myelogenous leukemia. Use of allotransplants for other indications continues to increase.
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  • Transcript of "Hematopoiesis - University of Medicine and Dentistry of New ..."

    1. 1. HEMOPOIESIS Arnold D Rubin, MD Cancer Institute of New Jersey Jan , 2009
    2. 2. HEMOPOIESIS <ul><li>Maintaining the optimal numbers of formed elements in the circulation: RBC for oxygen carrying,WBC for defense and platelets for liquidity without hemorrhage. </li></ul><ul><li>Elements in constant turnover: RBC-120 days,WBC-few days ( except for lymphocytes), platelets-short time. </li></ul><ul><li>Production must react to rapid changes in environment to provide homeostasis. </li></ul><ul><li>Only mature elements gain access to the circulation. </li></ul><ul><li>Plasticity. </li></ul>
    3. 3. How Can You Fulfill these Requirements? Only a mechanism involving stem cells could provide the plasticity and a life long rapid response to the ever changing inflammation, hemostatic and oxygen needs of the organism
    4. 4. CELL DEVELOPMENT STEM STEM STEM STEM STEM Hierarchic Stochastic Hemopoiesis Tumors
    5. 5. STEM CELLS <ul><li>A stem cell must be capable of self renewal and should not be “used up” </li></ul><ul><li>It must be capable of differentiating into a mature functioning cell </li></ul><ul><li>Committed stem cell can only differentiate into one type of cell </li></ul><ul><li>Pluripotential stem cell can differentiate into more than one type of cell </li></ul><ul><li>Totipotential stem cell can differentiate into any type of cell ( like an embryonic cell ) </li></ul>
    6. 6. Definition of a Hemopoietic Stem Cell 1 Repopulate the marrow of a lethally irradiated animal 2 Have the capacity for self renewal and expansion 3 Have the capacity to differentiate into all of the lymphomyeloid series
    7. 7. Replenish old or damaged tissue by limited proliferation and maturation Stem cell Differentiation leading to senescence and apoptosis THE FATE of the STEM CELL Mutation leading to neoplasia: ie failure to differentiate or undergo apoptosis
    8. 8. Genetics of Differentiation TF Silent HOX DNA RNA transcribed Translation into proteins to drive differentiation in to specific tissues Dormant or Dividing Stem Cells Epithelial Cell With differentiation mitotic potential is limited or ceases Cell ages and dies True stem cells are ageless
    9. 9. Identification and Function of Stem Cells Cell Surface Markers Functional Assays
    10. 10. Concept of Stem Cells Hemopoietic lethal radiation BM cells spleen colonies mouse Till- McCullogh Preparation CFU-S 8 day (committed) 12 day ( multipot.)
    11. 11. Maintainance of “Stemness” The length of the telomer determines the ability of the stem cell to keep from differentiating and aging. Thus, stem cells contain a lot of telomerase
    12. 12. M. Pera, ASH Education,2002
    13. 13. M. Pera, ASH Eduation,2002
    14. 14. M. Pera, ASH Education,2002
    15. 15. Hemopoietic Stem Cells- Surface Characteristics CD34-/lo CD34+ Sca-1+ Sca-1+ Lin- Lin- C-kit+ C-kit+ Low self renewel Multilineage Progenitors True stem cells Long term repopulation SKL Cells
    16. 16. Characteristics of Stem Cells Properties CD34++ Thy-1+ SSC-low c-kit+ HLADR- CD45RA- CD38- lin- CD13- CD33- in vitro Assays in vivo Assays LTC-IC Tx sheep in utero CAFC CFU-Blast SCID mice SCID-Hu Primates Human BMT Rh-123low FSC-low
    17. 17. Culture of Clonogenic Progenitors nutrients medium “ gamisch”: CSF’s lineage specific Interleukins semisolid suspension test cells colonies(CFU’s): Blast, GEMM, GM G,M,Meg, E,BFU etc
    18. 19. NOD-SCID Mouse Inject human stem cells CD38 CD45 Blood for FACS to show human cells grafted Assay for Human Stem Cells
    19. 20. Ontogeny of Hemopoiesis <ul><li>Primitive – transient in yolk sac, fetal Hb RBC -7.5 dpc (mouse) </li></ul><ul><li>Migration to fetal liver and spleen </li></ul><ul><li>By birth nearly all in bone marrow </li></ul><ul><li>Cell of origin hematogenic endothelium or hemangioblasts? </li></ul>
    20. 21. Orkin,S, Cell,2008
    21. 22. Orkin,S, Cell,2008
    22. 23. Other Cells and their Products Interact with Stem Cells
    23. 24. THE JACK SPRAT RIDDLE <ul><li>Marrow cells from the w/w mouse will not engraft a normal mouse </li></ul><ul><li>This mouse will accept a marrow graft from normal mice </li></ul><ul><li>The sl/sl mouse will not accept a marrow cell graft from normal or w/w mice, but its marrow cells will engraft normal and w/w mice </li></ul><ul><li>What is going on here? </li></ul>
    24. 25. The Steel Factor-Stem Cell Stromal Interaction Bone Marrow Stroma SLF SLF normal w/w sl/sl c-kit c-kit ( ( c-kit c-kit ( ( Stem Cells anemias
    25. 26. The Big Questions <ul><li>Infused marrow cells know to go directly to the bone marrow </li></ul><ul><li>No matter how many stem cells infused, the circulating mature cell numbers are “just right” </li></ul><ul><li>Pure CD34 positive stem cells are insufficient to restore a lethally irradiated bone marrow </li></ul><ul><li>WHY? HOW? </li></ul>
    26. 27. The Concept of a Hemopoietic NICHE <ul><li>BONE NICHE – Maintenance of long term quiescience of hemopoietic stem cells in appropiate numbers for blood cell homeostasis </li></ul><ul><li>VASCULAR NICHE – Proliferation of hemopoietic stem cells, their differentiation into mature blood cells and delivery to the circulation </li></ul>
    27. 28. Mutant Mice and the Bone Niche Runx-2 -/- mutant lacks osteoblast formation. Consequently no hemopoiesis is found in the bone marrow, hemopoiesis is present in the liver and yolk sac. cbfb -/- mutant lacks hemopoiesis in fetal liver Migration to bone marrow depends on vascular factors such as vegf and tel .
    28. 29. Orkin,S, Cell,2008
    29. 30. Copyright ©2006 American Society for Clinical Investigation Yin, T. et al. J. Clin. Invest. 2006;116:1195-1201 Hematopoiesis of bone cells and marrow stromal cells
    30. 31. Marrow Stroma HSC BMP Interaction HSC SC SCF c-kit Jagged1 Notch Ang1 Tie-2 Cadherin βcatenin VCAM integrin Osteopentin βintegrin Other Factors Affecting Interaction and Proliferation of HSC BMP Wnt Hedgehog Il-3 Il-6 TPO PTH
    31. 32. Copyright ©2005 American Society of Hematology. Copyright restrictions may apply. Taichman, R. S. Blood 2005;105:2631-2639 Figure 1. Model of support for hematopoietic stem cells by osteoblasts
    32. 33. Copyright ©2005 American Society of Hematology. Copyright restrictions may apply. Taichman, R. S. Blood 2005;105:2631-2639 Figure 2. Model of secreted and cell-associated factors produced by osteoblasts that influence HSCs
    33. 34. Copyright ©2005 American Society of Hematology. Copyright restrictions may apply. Taichman, R. S. Blood 2005;105:2631-2639 Figure 3. Model of HSC-OB adhesion-ligand pairs
    34. 35. Zhang,Nature,2003
    35. 36. Zhang,Nature,2003
    36. 37. The Vascular Niche <ul><li>More support for proliferation and differentiation of HPC’s by sinusoidal endothelial cells </li></ul><ul><li>Mobilization and homing of HPC’s </li></ul><ul><li>Backup in spleen when bone marrow is not functioning </li></ul>
    37. 38. Factors Involved in the Vascular Niche <ul><li>SDF-1 is a homing factor, inducing HSC’s to transendothelial migration mediated by E- and P-selectins. </li></ul><ul><li>HSC receptor is CXCR4, aided by Rac family </li></ul><ul><li>G-CSF increases SDF-1, elastase, cathepsin-G etc in circulation to facilitate mobilization </li></ul>
    38. 39. Copyright ©2006 American Society for Clinical Investigation Yin, T. et al. J. Clin. Invest. 2006;116:1195-1201 The osteoblastic and vascular niches in bone
    39. 40. Zhu,Oncogene,2002
    40. 41. Transcription Factors These factors play a vital role in both embryogenesis and lineage restricted differentiation of hemopoietic stem cells. Curiously most of these factors are subjected to mutations resulting in the development of hematologic malignancies. e.g. MLL, RUNX1,TEL/ETV6, SCL/tal1, LMO2 Detection is by knock-out and “knock-in” experiments in various model systems. MicroRNA’s, a new field, probably are also vital
    41. 42. Orkin, SH, Cell, 2008
    42. 43. Orkin,SH, Cell, 2008
    43. 44. Blood Cell Development Lympho-myeloid growth depends on a hierarchic array of clonogenic cells with widely varying potential for replication and differentiation
    44. 45. Hierarchy of Myeloid Cell Production S S gemm gemm GM GM GM GM Stromal Cell Macrophage T-cell CD34 + Sca + Lin - Thy-1 + ) ) ( ) ( L e s s d I v I s I o n M o r e m a t u r e IL’s CSF’s
    45. 46. Hemopoietic Cytokines Metcalf designed the system to demonstrate the existence and specificity of the various cytokines important in hemopoiesis Dr Metcalf
    46. 47. Culture of Clonogenic Progenitors nutrients medium “ gamisch”: CSF’s lineage specific Interleukins semisolid suspension test cells colonies(CFU’s): Blast, GEMM, GM G,M,Meg, E,BFU etc
    47. 49. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
    48. 50. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
    49. 51. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
    50. 52. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
    51. 53. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Metcalf, D. Blood 2008;111:485-491
    52. 54. Passegue,PNAS,03
    53. 55. Bruno,Mol & Cell Biol,2004
    54. 56. Stem Cell Transplantation(BMT) BMT can be used to replace a failed marrow or correct certain genetic defects. The adoptive immune system growing out of a BMT may be used to destroy hematologic malignancies resistant to chemotherapy BMT can be the vehicle to deliver a therapeutic gene in a viral vector. BMT may contain stem cells with multipotential plasticity for organ repair
    55. 57. ANNUAL NUMBERS OF BLOOD AND MARROW TRANSPLANTS WORLDWIDE 1970-2002 NUMBER OF TRANSPLANTS YEAR 1970 1975 1980 1985 1990 1995 Autologous Allogeneic 2000 1 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000
    56. 58. stem cells T cells stromal myeloid progenitors cancer cells other cells Bone Marrow Transplantation BMT is composed of disparate cells There are cells contributing to: maturing myeloid cells myeloid expansion support for myeloid growth immune activity GVHD reintroduction of malignant cells These may be conflicting
    57. 59. Sources of Stem Cells Source Bone Marrow Stimulated Blood 1/4-1/5 8-9 Cord Blood slightly increased 20-46 Fetal Liver/BM rich source similar to CB Content of Stem Cells relative to BM ----------- 21-30 Days to ANC Of 500 in vitro Expansion concentrated no faster than PB
    58. 60. Nonmyeloid Stem Cells in vitro expansion of CD34 + , CD45RA low CD71 low By IL-3, IL-6 SLF & EPO provides an expansion of : 5x for marrow 187x for UC blood 2125x for fetal liver More primitive by ontogeny yields stem cells more active in proliferation because telomeric DNA has not been lost yet.
    59. 61. Stem Cells from Sources Other than Bone Marrow <ul><li>Bood has 10-100 fold fewer stem cells as compared to marrow. But stimulation by cytotoxins &/or CSU’s results in peripheralization of CSF’s & to a lesser extent LTC-IC’s </li></ul><ul><li>Umbilical cord (UC) blood has more LTC-IC’s than marrow. But it takes 13-46 days for leukocyte recovery. </li></ul><ul><li>UC blood is easy to recover but limited in volume. Need >2X10 7 /kg nuc. cells </li></ul>
    60. 64. Adult Stem Cell Plasticity Hemic Stem cells Mesenchymal Stem Cells Liver Neurons, glial cells Cardiac Fibroblasts Osteoblasts Adipocytes Chondroblasts
    61. 65. C. M. Verfaillie, ASH Education, 2002
    62. 66. ex vivo Stem Cell Expansion <ul><li>Efficiency of harvest. At times this might be the only way to obtain sufficient cells. </li></ul><ul><li>As cells may be in synchronous cycle, gene transfer may be facilitated. </li></ul><ul><li>With appropriate cytokines, one could design grafts according to specifications. </li></ul><ul><li>This may be a means of obtaining grafts more likely to be free of tumor cells. </li></ul>
    63. 67. ex vivo Expansion of Stem Cells <ul><li>Incubate with stroma. Porcine stroma supports 5 fold expansion in 5 weeks. Maybe negative influence as well. </li></ul><ul><li>Incubate with mixtures of cytokines without stroma yield limited growth. There is no LTC-IC expansion. </li></ul><ul><li>Biorectors are very promising but only 10% of cells are responsible for growth. </li></ul><ul><li>No real success because niche can not be replicted </li></ul>
    64. 68. Retroviral Vectors Used to Transduce Stem Cells helper DNA gag pol env gag pol env vector viral RNA proteins helper virus RNA proteins empty virions RNA proteins vector in virions
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