1. XY XX 48 hrs Sacrifice, take marrow XX XY Short-term reconstituting XX blood cells Blood is XY Other organs have XY cells months Most strict definition of an HSC: ability to serially reconstitute hematopoiesis (HSC loses ability to do this after it has self-renewed several times) Serial transplantation to 2 nd ary host HSC home to marrow & are quiescent for up to 48 hrs

2. Krause et al, 2001 Cell 105:369 Plasticity of hematopoietic stem cells Circulating HSCs that have capacity to give rise to neurons, muscle (smooth, cardiac, skeletal), intestine, liver, skin, and lung tissue. Saul Sarkis’ lab at Hopkins performed serial long-term reconst. with one HSC Therapeutic implications… HSCs with ability to differentiate into epithelial cells target cells for gene therapy or source for organ reconstitution & repair FISH for Y chromosome Lung bronchi epithelium

3. Plasticity of (adult) hematopoietic stem cells From NIH Stem Cell Primer at http://www.nih.gov/news/stemcell/scireport.htm Hematopoietic stem cells may differentiate into: three major types of brain cells (neurons, oligodendrocytes, and astrocytes); skeletal muscle cells; cardiac muscle cells; and liver cells. Bone marrow stromal cells may differentiate into: cardiac muscle cells and skeletal muscle cells. Brain stem cells may differentiate into: blood cells and skeletal muscle cells

4. Cytokines play a major role in influencing the direction in which differentiation proceeds… Support the survival and/or proliferation of certain subsets of cells in the differentiation pool Exert a wide variety of biological functions on various cell types (e.g., IL- 6: B cell maturation, T cells, hematopoietic progenitor cells, hepatocytes, neuronal cells). Can be redundant & overlapping

5. Cytokines Progenitor cells require a minimum of 2 cytokines to grow effectively – express receptors for multiple cytokines COMBINATORIAL EFFECT Committed precursor cells can grow in response to a single cytokine (progressive “focusing” of cytokines receptors on cells as cells move toward commitment to a terminally differentiated cell type) UNILATERAL EFFECT TPO

6. 2 examples of common clinically utilized cytokines which affect terminal differentiation: G-CSF: A) sustains myelopoiesis – expansion of committed myeloid precursor cells. Also, increases sensitivity of cycling stem cells to other cytokines and increases their entry into cell cycle. Used in patients who are neutropenic after chemotherapy to speed recovery of normal granulocyte counts. B) Induces mobilization of CD34 + stem cells prior to HSC transplantation. Erythropoietin: sustains erythropoiesis. Treatment of chemotherapy- induced anemia in cancer patients, other anemias. Illegal blood doping in athletes. Blood doping is bad, Mkay ?

7. Cytokines signal through specific receptors Most cytokine receptors consist of a multisubunit protein complex - unique and specific ligand binding subunit - signal transducing subunit, which may be structurally similar to other members of the cytokine receptor superfamily. (ligand-binding) (signal-transducing)

8. Modular nature of cytokine receptors: For a number of receptors, proximal cytoplasmic region is sufficient for proliferation, distal sequences are required for differentiation Most cytokine receptors lack intrinsic tyrosine kinase activity, and signal through cytoplasmic partners: They must recruit cellular tyrosine kinases to mediate their downstream signal transduction pathways. Several different pathways, including shc/ras, shc/PI3K, JAK/STAT pathway.

9. 1)Interaction of ligand with receptor leads to dimerization of receptor and recruitment & activation of Janus kinases (JAKs) 2)JAKs phosphorylate receptor’s cytoplasmic domain on tyrosine (Y) residues 3)Phosphorylated tyrosine residues serve as docking sites for STATs 4)JAKS phosphorylate STATs (signal transducers and activators of transcription) 5)STATs dissociate from receptor, dimerize, and translocate to nucleus 6)STATs activate transcription of genes (e.g., those involved in terminal differentiation) JAK

10. Myeloid progenitor

11. Stat-3 activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation McLemore et. al., Immunity, 14:193-204 (2001) G-CSF – the principal hematopoietic cytokine regulating the production of neutrophils Effects of G-CSF are mediated solely through its interaction with the G-CSFR. STAT-3 is the principal STAT protein activated by G-CSF, with STAT-5 and STAT-1 to a lesser degree… what is the contribution of STAT-3 to the signaling function of G-CSF??

12. Background G-CSF induces (activates) Stat3 Mice lacking G-CSF or G-CSF receptor do form neutrophils, though in significantly reduced numbers (severe neutropenia)… accumulate immature myeloid precursors in marrow, and granulocyte differentiation is severely impaired Expression of DN-Stat3 (which can’t bind DNA) blocks G-CSF-mediated differentiation in cell lines… get sustained G-CSF mediated proliferation without granulocytic differentiation This group had previously generated a targeted mutation of G-CSFR in mice with the distal 98 AAs deleted (deleting 3 of 4 Ys), leaving only one terminal Y at AA 715 intact. These mice had normal granulopoiesis and activated STAT-3 in response to G-CSF.

13. wt (813aa) d715 d715F Y F Y Y Generates neutrophils, activates Stat3 G-CSFR MUTANT MICE USED IN McLEMORE STUDY Generates neutrophils, activates Stat3 Hypothesized that this would not activate Stat 3 (can’t dock at phenylalanine)

14. 1)Interaction of ligand with receptor leads to activation of Janus kinases (JAKs) 2)JAKs phosphorylate receptor’s cytoplasmic domain on tyrosine (Y) residues 3)Phosphorylated tyrosine residues serve as docking sites for STATs 4)STATs (signal transducers and activators of transcription) are phosphorylated by JAKs 5)STATs dissociate from receptor, dimerize, and translocate to nucleus 6)STATs activate transcription of genes (e.g., those involved in terminal differentiation) JAK

15. Assay for activation of STAT-3: EMSA with radioactive DNA oligo specifically bound by STAT-3 in nuclear extracts Assay for activation of STAT-5 BM isolated from mice given 10 min. treatment w/ cytokine (G-CSF in comparison to IL-6) Mice had 7 days treatment w/ G-CSF, BM isolated 4 hrs. after last injection “ “ (G-CSF in comparison to IL-3)

16. In vivo response to G-CSF: D715F mice had normal promyelocytes, increased myelocytes, and decreased bands and neutrophils

17. Example of differentiation from the precursor stage onward: myeloblast to granulocyte (neutrophil maturation) Increasing development of granuoles (antibacterial and phagocytic) Increasing phagocytic function – pseudopodia extend around microorganisms and fuse to form a phagosome into which granuole contents are released 2.6 X 10 9 cells/kg in mitotic pool 9 X 10 9 cells/kg in post-mitotic pool Expansion of cell number occurs as cells in the mitotic or proliferative pool replicate The post-mitotic pool can no longer divide but continues to mature into terminally differentiated cells In the setting of infection or stress, maturation time may be shortened, divisions may be skipped, and cells may be released into the bloodstream earlier Terminal cell division

18. Hematopoietic progenitor assays – ability of BM (progenitor cells within) to form differentiated colonies in vitro

19. Expression of constitutively activated Stat3 (Stat3C – contains 2 Cys in its SH2 domain, which causes spontaneous dimerization) restores some G-CSF responsiveness (induction of colonies) Can STAT3 rescue d715F mutants from block in diff’tn? MSCV – control “empty vector”

20. G-CSF induced differentiation is enhanced by Stat3C transfection of mutant mice bone marrow cells Neutrophils & bands Cultured 7 days in presence of G-CSF MSCV= murine stem cell virus (empty vector)

21. Conclusions: Stat activation, particularly of Stat3 and Stat5, play an important role in myeloid differentiation and survival… Common theme that mutant cytokine receptors lacking Stat recruitment sites fail to induce differentiation

22. Transcription factors

23. Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein  (C/EBP  Nakajima and Ihle, BLOOD, 98, 897-905 (2001)  Background: C/EBP  is a (granulocyte) lineage-specific transcription factor upregulated at the promyelocyte or myelocyte stage and expressed thereafter. KO mice revealed a critical role in mid-late stage of granulocytic differentiation (produce mature granulocytes but they are morphologically and functionally abnormal). Using 32Dc13 cells – an IL-3 dependent murine myeloid line that can be induced to differentiate to neutrophils or macrophages using G-CSF or GM-CSF, respectively.

25. Nakajima, Blood 2001 G-CSF stimulation Northern results agree with those of Western blots NFS60 do not differentiate, but rather proliferate in response to G-CSF IL-3 negative control (don’t differentiate to granulocytes)

26. Nakajima, Blood 2001 C/EBP  Stable transfectant Also showed constitutive MPO expression in stable transfectant

27. Nakajima, Blood 2001 98:897 Is Tyr 703 (same as Y at 715 previous study) of G- CSFR important for induction of C/EBP  by G-CSF? YES… Might suggest a role for STAT-3

28. Lacks activation domain Using wt G-CSFR, but mutant STAT3 BUT, expression of C/EBP  and MPO in DN- STAT3 transfected cells indicates normal induction Morphological features of DN-STAT3 transfected cells show differentiation block Check to see if STAT-3 activates C/EBP  …

29. Therefore, C/EBP  and STAT3 work in different pathways and C/EBP  is regulated by a signaling molecule other than STAT-3 Previous mutant mice were generated in which the cytoplasmic domain of G-CSFR was replaced with that of EPO-R, and had no apparent defect in lineage commitment… suggests that signaling to induce C/EBP  generated from G-CSFR can be replaced by Epo signaling and should be a common pathway used by both Epo and G-CSF.