Study of the role of Ctbp2, Mrps6 and Ccr7 intrinsic regulatory pathways in murine hematopoietic stem cells

Overespressione di Ctbp2 e Mrps6 e loro ruolo nell’espansione di cellule staminali ematopoietiche Il fattore trascrizionale a funzione repressoria Ctbp2 e il gene di nuova scoperta Mrps6 sono stati recentemente identificati per essere maggiormente espressi nel compartimento staminale rispetto a quello ricco in progenitori. Il knockdown di Ctbp2 e Mrps6 in embrioni di Zebrafish porta ad una riduzione della produzione di sangue e dell’espressione di marker ematopoietici, quali scl, gata1, hbae1 and lcp1. Per valutare il ruolo di Ctbp2 e Mrps6 in un modello di mammifero abbiamo trasferito il cDNA di questi geni in cellule staminali ematopoietiche murine, cellule di midollo osseo cosiddette lineage negative (Lin-), tramite una trasduzione retrovirale. Abbiamo usato le cellule così modificate, che overesprimono il nostro gene di interesse, per trapianti competitive di midollo osseo in topi irradiati in modo letale, allo scopo di valutare l’attecchimento del trapianto. In vitro abbiamo valutato la capacità differenziativo di queste cellule mediante il saggio di Colony Forming Cells (CFCs) e il loro fenotipo tramite tecniche di citofluorimetria. L’espressione forzata di Ctbp2 e Mrps6 in cellule Lin- murine porta ad una significativa riduzione dell’attecchimento e a un ridotto sviluppo di CFCs, comparando I risultati dati da cellule trasdotte col vettore vuoto. Invece l’overespressione Di Hoxb4, che è ben noto in letteratura avere un ruolo nell’espansione di cellule staminali ematopoietiche, mostra una incrementata capacità di attecchimento. Ctbp2 e Mrps6 sembrano quindi avere un ruolo chiave nel mantenimento/differenziazione di cellule staminali ematopoietiche conservato in specie diverse.

Overexpression of Ctbp2 and Mrps6 and their role in hematopoietic stem cells expansion The transcriptional co-repressor Ctbp2 [2] and the novel Mrps6 gene [3] have recently been identified to be more highly expressed in the human hematopoietic stem cell rich compartment versus human committed progenitor cells [1]. Ctbp2 and Mrps6 knockdown in zebrafish embryos resulted in decreased blood formation and reduced expression of hematopoietic markers, scl, gata1, hbae1 and lcp1. To evaluate the role of Ctbp2 and Mrps6 in a mammalian model, we transferred the cDNA of these genes in murine hematopoietic stem cells, lineage depleted (Lin-) bone marrow cells, by retroviral transduction [4]. We used the cells, overexpressing our gene of interest, for a competitive repopulation assay into lethally irradiated mice to investigate the transplantation outcome. In vitro we evaluated the cells differentiation capacity by Colony Forming Cells assay (CFCs) and their phenotype by FACS. Forced expression of Ctbp2 and Mrps6 in mouse Lin- cells leads to a significant lower engraftment as well as to a decreased number of CFCs, compared to cells transduced with the control vector. Instead overexpression of Hoxb4 [5], which is known to affect expansion of hematopoietic stem cells, showed an increased engraftment potential. Ctbp2 and Mrps6 thus seem to have a key role in maintenance/differentiation of hematopoietic stem cells, conserved among different species. Inducible overexpression of Ctbp2 and Rassf8 using murine embryonic stem cells We showed that constitutive overexpression of Ctbp2 and Rassf8 could inhibit differentiation as the number of CFCs is decreased. In this way it is possible that hematopoietic stem cells engraft perhaps even expand. However, due to a block of differentiation, we may incorrectly conclude that Ctbp2 and Rassf8 prevents hematopoietic stem cells engraftment. Therefore it is necessary to evaluate overexpression in an inducible way. We chose the W41/W41 complementation strategy. Mouse embryonic stem cells, kindly provided by Rudolf Jaenisch lab (Massachusetts Institute of Technology) [6] have been transfected with a plasmid that contains the gene of interest. After the integration, the gene will be under the control of a tetracycline promoter. Subsequently these modified embryonic stem cells have been injected in the blastocyst of W41/W41 mice that have a mutation in the ckit gene, which causes a severe hematopoietic defect [7]. Because of this mutation, the hematopoietic system of the transgenic progeny is almost completely donor derived [8]. The fetal liver cells, consisting of 98% donor derived, are under in vitro evaluation. The expression of the gene can be induced in vivo or in vitro using doxycycline. In vitro tests consist in evaluation of cell expansion, analysis of the phenotype by flow cytometry and CFC assays. In vivo we will perform competitive repopulation assays, transplanting the fetal liver cells into lethally irradiated mice and evaluating the engraftment outcome. In this way we can show whether Ctbp2 and/or Rassf8 can expand the hematopoietic stem cells pool and we can investigate the role of these genes during the hematopoietic system development. References [1] Eckfeldt CE et al. PLos Biol. e254:1-10 (2005) [2] Spyer M, Allday MJ Cell Cycle 5:5, 530-537 (2006) [3] Sultan M et al. Genome Biol. 8(5):R91 (2007) [4] Rizo A et al. Blood 1;111(5):2621-30 (2008) [5] Antonchuk J et al. Cell 5;109(1):39-45 (2002) [6] Beard C et al. Genesis 44:23-28 (2006) [7] Eckardt S, McLaughlin KJ Methods in Molecular Biology, vol. 430: Hematopoietic Stem Cell Protocols, c. 14 [8] Jansson L, Larsson J Blood, online 28 oct 2009