Early Blood Cell Formation in sickness and health, till death do us part Kees-Jan Pronk Sektion för Immunologi& Hematopoietisk Stamcell Laboratoriet, Medicinska fakulteten, Lunds Universitet Barn- & Ungdomssjukhuset, Lund Mentor: David Bryder Co-mentor: Sten Eirik Jacobsen VPH möte Stockholm 100204. Erytrocytens dag.
Vad händer i the black box? under fysiologiska betingelser, hos sjuka och vid åldrandet Mogen blodcellsbildning allt ifrån en cell HSC (hematopoietiska stamcellen)
Vad händer i the black box? (immature hematopoiesis) HSC (hematopoetiska stamcellen) Multi-potent Oligo-potent/ Oligo-primed Uni-potent
Stamcellskonceptet Olika funktioner självförnyelse (identisk kopia). differentiering i >1 typ av dotterceller Kan (funktionellt) ersätta ett organ. reservdel Olika typer embryonala stamceller cancer stamceller vuxna (somatiska) stamceller
Toti-, pluri-, multi-, bi-, unipotent home.earthlink.net/~zaptos/website/media/differentiation.jpg www.nih.gov/news/stemcell/primer.htm
Cancer stamceller Varför är cancerstamceller viktiga? 1. Infrekventa celler. 2. Ansvarar för uppkomsten och underhåll av tumören (vissa/alla?). 3. Ofta terapiresistenta
Cancer stamceller Illustrerad vetenskap, nr 8 2007 Efter celldelningen får inte bara den nya stamcellen utan också mellanformerna och senare de färdiga vävnadscellerna skador - i form av aneuploidi och/eller mutationer
Embryonala stamceller www.nih.gov/news/stemcell/primer.htm ny DNA Nuclear transfer ( --- Kloning) egen DNA ES cell culture
Ian Wilnut och Dolly (1997) Embryonala stamceller
ips - induced Pluripotent Stem cells Illustrerad vetenskap, Nr 14, 2008. ips Även i blodsystemet: Treatment of sickle cell anemia mouse model with ips cells generated from autologous skin. Hanna J, et al. Science 2007
Overview of included studies Article I (TNF) Hematopoietic stem cell (HSC) Most studied and well defined organ restricted stem cell Resides in adult bone marrow (BM) in HSC niches Resides in BM at low frequencies (1/10.000-20.000) Can functionally repopulate tissue of origin Potential to produce all blood cell lineages. Life-long!!! Widely used in clinical practice Responsible for cellular output of 5.000.000 cells/sec in man Article II (Aging) Article III&IV (subfractionation) 13.500.000.000 blodceller under denna föreläsning
Article III&IV Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Pronk, C.J.*, Rossi, D.J.*, Mansson, R., Attema, J.L., Norddahl, G.L., Chan, C.K., Sigvardsson, M., Weissman, I.L., and Bryder, D. Deciphering developmental stages of adult myelopoiesis. Pronk, C.J., Attema, J., Rossi, D.J., Sigvardsson, M., and Bryder, D.
Articles III&IV: lite bakgrund Articles III + IV study the developmental stages of hematopoiesis and regulators involved in these processes Why is this important? 1. To better understand normal biology 2. To better understand abnormal blood cell production, f.i. Aging: lineage skewing Cancer: block in differentiation
Articles III&IV: some background. The black box.. Bryder et al. 06
Articles III&IV: experimental assay FACS (fluorescent assisted cell sorting) 10 5 26.3 10 4 10 3 0 73.6 Sort green cells 0 10 3 10 4 10 5 Run samples for analysis Functional evaluation Gene expression evaluation Etc.
Articles III&IV: Immunophenotypic analysis of myeloerythroid progenitors MkP: Megakaryocytic progenitor Pre GM: pre Granulocyte/Macrophage GMP Granulocyte/macrophage progenitor Pre MegE pre Megakaryocyte/Erythrocyte Pre CFU-E pre Colony Forming Unit - Erythrocyte CFU-E: Colony Forming Unit - Erythrocyte
Articles III&IV: Functional evalutation In vitro investigation of erythroid, megakaryocytic, granulocytic and monocytic lineage potentials In vivo investigation of erythroid, lineage potentials
Articles III&IV: Gene expression analysis Population based or single cell evaluation of gene activity, both global gene profile and a selected set of genes
Articles III&IV: Some conclusions 1. Phenotypic and functional identification of Pre MegE, Pre CFU-E and Pre GM cells 2. No evidence for the existence of the previously identified CMP 3. Hierarchical and lineage relationship of newly and previous identified cellular subsets 4. Closer developmental relationship of the innate ( G/M ) and adaptive ( lymphoid ) immune system than previously assumed
Slamf1+ CD34 Endoglin+ Flt3 Composite model of early hematopoiesis Lineage- (Ter119- B220- CD4- CD8- C3- CD11b- Gr1-) KLS Itga2b?? LT- HSC Ikaros-/Pu.1- ST- HSC? Pu.1- Gata1+? Itga2b- Endoglin+/- pre MegE Slamf1+ Itga2b+ MkP* Slamf1+ Endoglin+ pre CFU-E CD71+/- Meg* CFU-E Slamf1- KLS- Ikaros+/Pu.1+ VCAM+ LMPP/ GMLP Pu.1+ Gata1- Endoglin+/- pre GM Slamf1- FcgR+ Flt3- GMP K +/- LS +/- IL7Ra+ VCAM- CLP B prog NK prog T prog Proliferation capa- Multi- city potentiality
Hur kan vi nu använda dessa resultat? Exempel i den röda linjen FSC-A 4000 3000 2000 1000 0 0 102 103 104 105 Lineage 10 5 10 4 ckit 10 3 0 0 103 104 105 Sca1 CD150 10 5 10 4 10 3 0 0 102 103 10 4 105 CD41 CD105 10 5 10 4 10 3 0 0 10 3 10 4 10 5 CD150 ii. i. CD71 10 5 10 4 10 3 0 5 iii. iv. 10 10 4 v. 0 10 3 10 4 10 5 Ter119 CD71 10 3 0 vi. vii. 0 10 3 104 10 5 Ter119 More immature... pre CFU-E* Pro EB Late Baso&Chro EB i. ii. iii. iv. v. vi. vii. pre MegE* CFU-E Baso EB Orth EB Retikulocyt Kartläggning av hela den röda linjen Erythrocyt
Hur kan vi nu använda dessa resultat? Exempel i den röda linjen Gamla möss 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Young Mer myeloid + megakaryocyt GM Old 0.12 0.10 0.08 0.06 0.04 0.02 0.00 Young MkP Äldre människor - Anemi - Blodpropp sjukdomar - Myeloida sjukdomar (MDS, leukemier) Old Frequency in bone marrow (%) 0.06 0.05 0.04 0.03 0.02 0.01 0.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 Pre CFU-E Young Basophilic - Rel mindre lymfoida leukemier jmf barn (+dålig Ak-svar) + samma translokationer olika leukemier hos barn jmf vuxna!! Young Mindre erytroid Old Old 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 CFU-E Young Old Late Baso- & Chromatophilic Young Old Proerythroblasts 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 Young Old Orthochromatic Normoblasts 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Young Old studier i människor
Hur kan vi nu använda dessa resultat? Exempel i den röda linjen Mitokondrial dysfunktion - prematurt åldrande Block i utmognaden inblandning av mitokondrier i leukemier/erytrocytära sjkd??
Hur kan vi nu använda dessa resultat? Exempel i den röda linjen TEC? Block i utmognaden
Sista slide Min framtidsförhoppning vid en abnormal röd linje (erytrocytär sjkd) inga kroniska transfusioner, utan: bot, eller expansion av kroppens egna celler TACK!!