Bone marrow examination | EPFL Graph Search

Bone marrow examination refers to the pathologic analysis of samples of bone marrow obtained by bone marrow biopsy (often called trephine biopsy) and bone marrow aspiration. Bone marrow examination is used in the diagnosis of a number of conditions, including leukemia, multiple myeloma, lymphoma, anemia, and pancytopenia. The bone marrow produces the cellular elements of the blood, including platelets, red blood cells and white blood cells. While much information can be gleaned by testing the blood itself (drawn from a vein by phlebotomy), it is sometimes necessary to examine the source of the blood cells in the bone marrow to obtain more information on hematopoiesis; this is the role of bone marrow aspiration and biopsy. Components of the procedure Bone marrow samples can be obtained by aspiration and trephine biopsy. Sometimes, a bone marrow examination will include both an aspirate and a biopsy. The aspirate yields semi-liquid bone marrow, which can be examined by a

Investigation of transcriptional partners and genes downstream of Foxn1

Zineb Bekkali

The thymus is the second fundamental organ of the immune system after the bone marrow; it is the essential for T cell maturation and repertoire selection. The function of the thymus critically depends on the thymic epithelium, which is structured in two distinct regions: The cortex and the medulla. The knowledge about molecular mechanisms involved in thymus development is limited. However, some evidences suggest that the Hox, Pax, Six, Eya, Gcm2, FGFs and Foxn1 are key players in thymus organogenesis. Foxn1 is essential for thymic epithelial cell development but its exact role is still unknown. The null mutation in the transcription factor Foxn1 generates the nude phenotype of athymia and hairlessness. The aim of our laboratory is to better understand thymus development and specially focuses on the Foxn1 function. The aim of this project is then to investigate target genes and transcription partners of Foxn1 as a transcriptional factor. As a transcription factor, Foxn1 protein should be able to bind with DNA but should also interact with other proteins such as cofactors in order to activate its target genes. At present, the transcriptional partners of Foxn1 are completely unknown in the literature and the proteinprotein interaction databases. The experimental approach proposed here is the standard method for identification of unknown transcriptional partners: Immunoprecipitation (IP) followed by a mass spectrometry analysis. The list of protein candidates can then be validated by Western blotting. We identified the right antibodies to use for this procedure and optimised protocols for the IP. We also performed a mass spectrometry analysis that was not conclusive, however, the platform is now established to complete this analysis. Only little is known about genes up- or down-stream Foxn1. Some genes such as FgfR2IIIb, DLL4 and CCL25 are believed to be up-regulated by Foxn1. Our laboratory has generated a revertible hypomorphic allele of Foxn1 and have used this to obtain a series of six allelic combinations at Foxn1. These mice, expressing different levels of Foxn1, are a very useful tool in order to investigate target genes of Foxn1. In particular, we wish to investigate whether different Foxn1 targets are activated at different Foxn1 levels. 6 To reach this objective, the proposed approach is the comparison of gene expression profiles of thymic epithelial and mesenchymal cells of the allelic series at two different time points E13.5 and E15.5 using a Solexa tool called SAGE (Serial Analysis of Gene Expression). As preliminary analysis before the global gene expression analysis, we have determined expression profiles of several genes across the allelic series by quantitative PCR. Our findings suggest that DLL4, CCL25, FgfR2IIIb, FGF10 and possible Pax1 are direct or indirect targets of Foxn1 activated at different levels of Foxn1. DLL4 and CCL25 seem to be involved in lymphocyte attraction and maturation. FgfR2IIIb appears to be important for thymic epithelium differentiation and development by increasing the sensitivity of epithelial cells to FGF10 signalling. Finally, Pax1 is possibly required downstream of Foxn1 for terminal differentiation of cortical thymic epithelial cells

2009

Characterization of STAT1-/- mice & Investigation of IFN alpha/beta production in murine bone marrow in response to injury

Yulia Kirpicheva

The hematopoietic system is a regenerative tissue with high cell turnover. Normal homeostasis of the hematopoietic system is insured by hematopoietic stem cells (HSCs). The important features of HSCs are the capacity to self-renew and multipotency. Multipotent HSCs perpetuate through life and are able to form all differentiated cell lineages of the blood. Most HSCs with long-term self-renewal capacity are quiescent and divide infrequently. However quiescent HSCs can be activated in response to injury. The signals responsible for the activation of quiescent HSCs are poorly investigated. The Trumpp laboratory has recently identified a role for IFNα in activation of quiescent HSCs in vivo. Activation of HSCs by IFNα involves increased phosphorylation of STAT1. Therefore the role of STAT1 in the homeostasis of the hematopoietic system was investigated by characterization of the hematopoietic system in STAT1-/- mice. A 2-fold decrease in short-term HSC (ST-HSCs) and multipotent progenitors (MPPs) was observed in the bone marrow of STAT1-/- mice compared to C57Bl/6 (wild type) mice. Analysis of progenitors of HSCs also revealed a small increase in common myeloid progenitors (CMPs) and a small decrease in common lymphoid progenitors (CLPs) in the bone marrow of STAT1-/- mice. These results indicate that STAT1 play a role in the homeostasis of ST-HSCs and MPPs in bone marrow. Functional analysis of STAT1-/- LT-HSCs using in vivo long-term reconstitution assay suggests that STAT1-/- LT-HSCs can reconstitute lethally irradiated mice. Nevertheless further investigations are necessary to examine whether STAT1 plays a role in HSCs self-renewal. The unexpected role of IFNα in activation of quiescent HSCs leads to the hypothesis that endogenously produced IFNα might be important for activation of HSCs in response to injury. To address this we generated the mouse model MxCre;R26REYFP to monitor endogenous IFNα/β production in the bone marrow after injury. Our results suggest that IFNα/β is produced in response to 5-FU induced myeloid suppression and G-CSF treatment in vivo. The role of IFNα/β during the recovery phase after 5-FU induced myeloid suppression was investigated using IFNAR-/- mice. These results suggest that endogenous IFNα/β might be important for the mobilization of HSCs into the spleen induced in response to 5-FU

2008

Characterization of the paracrine stimulation potential of human CAP cells for cardiac repair

Xavier Adam

According to the last WHO numbers, cardiac diseases remain the first cause of death in the world. Cell therapy represents a promising approach to restore heart function. Cardiac derived adherent proliferation (CAP) cells were shown to support the heart regeneration by unexplained mechanisms when injected in a mouse myocarditis model. Not differentiating themselves in cardiomyocytes and expressing cellular markers for angiogenesis, they are now supposed to have a paracrine effect on heart resident cell populations. In the present work, this paracrine effect had to be characterized on a model cell line. A paracrine effect test system based the P19Cl6 teratocarcinoma cell line had to be established and used to carry out a first screening of paracrine interactions on CAP cells and mesenchymal stem cells (MSC) from bone marrow as reference. Initially, critical parameter of the test system had to be adjusted. Specific marker must be identified and their detection had to be proven possible in the designed test system. Suitable serum lots (k,g) have been identified on the base of growth rates and cardiac differentiation potential (beating activity, Gata-4, Myl2, Mef2c gene expression) using standard protocol (ɑMEM, 1% DMSO). Pretreatment of P19Cl6 with 10 [micro]M 5-azacytidine substantially raised the response to known potent cardiomyogenic inductors (DMSO, Oxytocin). Beating activity was remarkedly elevated, as well as Gata4 (5 to 300 fold), Myl2 (2 to 300 fold), and MEF2C (3 to 4 fold) expression induced compared to non-treated culture. Troponin I ELISA-(2 to 5 fold) and Connexin 43 imunnohistochemistry confirmed the reliable induction of cardiac differentiation in pretreated culture. Based on those results, the test system was conducted using 5-azacytidine pretreated P19Cl6 cells and explored their reactions to different direct (cell to cell in coculture) and indirect (secreted factors in preconditioned medium) interactions with CAP cells or MSC. Since no reference setup existed, several coculture compositions with fixed total cell number (10-90%) or fixed P19Cl6 cell number (10-1000%) were tested, as well as exposition to differently conditioned media (24h, 72h) in different dilutions (100%, 50%, 33%). Beating activity was almost never observed. However, CAP cell experiments (n=6) showed a significant increase in P19Cl6 GATA-4 expression compared to control P19Cl6 (3.3 fold). This expression was also increased in MSC analyses (n=4) but in small amount (1.8 fold). Screening of CAP and MSC interactions led to the identification of active/effective compositions and dilutions. In addition, aza pretreatment proved itself enhancing the sensitivity of the test system. To sum up, the results suggested that CAP cells as wells as MSC have a paracrine stimulation potential and gave starting points for deeper analyses.

2011