The focus of our laboratory is to understand the molecular determinants underlying cell reprogramming and hematopoietic specification. In humans, the multiple differentiated cell states are normally stable and inherited through cell division. Under certain conditions, cell fate can, however, be modified or reversed. Cell reprogramming can be achieved experimentally in different ways, including nuclear transfer, cell fusion or expression of transcription factors. The emergent ability to directly reprogram somatic cells into desired hematopoietic cell-types is opening avenues to the discovery of new therapies for immune and blood diseases. Our approach focuses on Hematopoietic Stem Cells (HSCs) for their remarkable regenerative potential and Dendritic Cells (DCs) as key mediators of immunity.

 

Aims:

• To understand at the molecular level how hematopoietic cellular identities are specified during development employing cellular reprogramming

• To use this knowledge to allow the generation of patient-specific hematopoietic and immune cells for regenerative medicine and immunotherapy

 

Cell Reprogramming

Epigenetics

Hematopoietic stem cell

Dendritic cell

Immunotherapy

 

 

In humans, the 200 differentiated cell states are normally stable and inherited through cell division. Under certain conditions, cell fate can, however, be modified or reversed. Epigenetic reprogramming can be achieved experimentally in different ways, including through nuclear transfer, cell fusion, or the forced expression of transcription factors. The ability to reprogram or program differentiated cells into desired cell types is opening avenues to the discovery of new therapies for hematological disease. The goal of my laboratory is to understand how hematopoietic cellular identities, especially hematopoietic stem cells, are specified during development, and to use this knowledge to manipulate blood cells for regenerative medicine and cell therapy. To explore this aim, we use a variety of approaches, including genome-wide gene expression and chromatin profiling, cellular reprogramming and programming through gene transduction, computational analyses, directed differentiation of mouse embryonic stem cells, gene knockdown, mouse genetics and cellular transplantation.

2 - Programming, Reprogramming and Developmental Biology

We employ two complementary approaches to investigate the establishment of cellular identity: to define the minimal intrinsic determinants of hematopoietic cell fates, we screen for factors that re-create the generation of these unique cell identities from fibroblasts. This approach leads to the identification of master transcription factor minimal networks, cell surface phenotypes and gene expression signatures of induced cell fate. We then ask whether a similar logic applies to the specification of hematopoietic cell types during mouse embryogenesis and use this information to uncover general mechanisms of blood cell specification. We have previously shown the conversion of differentiated fibroblasts to hemogenic cells that undergo endothelial-to-hematopoietic transition and acquire hematopoietic progenitor activity. We are employing this induction in the human system to delve into the step-wise mechanistic regulation of the establishment of hematopoietic stem cell identity. We now extend our studies to the specification of mature hematopoietic cell-types and broader types of reprogramming. The minimal networks identified by direct induction of hematopoietic cell-types will be instrumental to elucidate the establishment of different cell identities and the exclusion of alternative cell fates throughout development.

3 - Development of New Therapies

Alongside with the understanding of the basic biology of hematopoietic specification, we aim to apply our findings to the treatment of human diseases. We intend to use human programmed cells in the clinic for cell therapy. In addition, we will use these new sources of patient-specific cells for the discovery of new therapies and drugs using chemical screens and for mechanistic studies of hematological disease. We believe that by combining classical cell biology and genetic approaches with current technologies we will enhance the knowledge of blood specification. This understanding can ultimately lead to the generation of patient-specific blood products for cell replacement therapy as well as for the discovery of novel chemicals for therapeutic intervention.

 

Luis Galán Palma

Cláudia Raquel Marques

Monika Małż

Julia Zdzieszyńska

Rigveda Bhave

Kritika Shaiv

Andreia Gomes

Cláudia Azenha

Tânia Barata

Raquel Santos

Piccolo F.M., Pereira C.F., Cantone I., Brown K., Tsubouchi T., Soza-Ried J., Merkenschlager M., Fisher A.G. (2011). Using heterokaryons to understand pluripotency and reprogramming. Phil. Trans. R. Soc. 366, 2060-5

Pereira C.F (2011). Epigenetic events underlying somatic cell reprogramming: insights from heterokaryons. LAP LAMBERT Academic Publishing ISBN: 3846537144.

Schnetz, M.P., Handoko, L., Akhtar-Zaidi, B., Bartels, C.F., Pereira, C.F., Fisher, A.G., Adams, D.J., Flicek, P., Crawford, G.E., Laframboise, T., Tesar, P., Wei, C.L., Scacheri, P.C. (2010). CHD7 targets active enhancer elements to modulate ES cell-specific gene expression. Plos Genet. 15, e1001023.

Pereira, C.F., Piccolo, F.M., Tsubouchi, T., Sauer, S., Ryan, N.K., Bruno, L., Landeira, D., Santos, J., Banito, A., Gil, J., Koseki, H., Merkenschlager, M., Fisher, A.G. (2010). ESCs require PRC2 to direct the successful reprogramming of differentiated cells towards pluripotency. Cell Stem Cell. 6, 547-556. ˥Recommended by the Faculty of 1000.

Kanhere, A., Viiri, K., Araújo, C.C., Rasaiyaah, J., Bouwman, R.D., Whyte, W.A., Pereira, C.F., Brookes, E., Walker, K., Bell, G.W., Pombo, A., Fisher, A.G., Young, R.A., Jenner, R.G. (2010) Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2. Mol Cell. 5, 675-88. ˥Recommended by the Faculty of 1000.

Landeira, D., Sauer, S., Poot, R., Dvorkina, M., Mazzarella, L., Jørgensen, H.F., Pereira, C.F., Leleu, M., Piccolo, F.M., Spivakov, M., et al. (2010). JARID2 is a PRC2 component in ES cells required for PRC1 and RNA Pol II recruitment at developmental regulator genes and multi-lineage differentiation. Nat. Cell Biol. , 6, 618-24.

Santos, J. *, Pereira, C.F. *, Di-Gregorio, A., Spruce, T., Alder, O., Rodriguez, T., Azuara, V., Merkenschlager, M., and Fisher, A.G. (2010). Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo. Epigenetics Chromatin 3, 1. *equal contributors.

Pereira, C.F., Fisher, A.G. (2009) Heterokaryon-based reprogramming for pluripotency. Curr. Protoc. Stem Cell Biol. Chapter 4:Unit 4B.1.

Savarese, F., Dávila, A., Nechanitzky, R., De La Rosa-Velazquez, I., Pereira, C.F., Engelke, R., Takahashi, K., Jenuwein, T., Kohwi-Shigematsu, T., Fisher, A.G., Grosschedl, R. (2009) Satb1 and Satb2 regulate embryonic stem cell differentiation and Nanog expression. Genes Dev. 22, 2625-38.

Banito A., Rashid S.T., Acosta J.C., Li S., Pereira C.F., Geti I., Pinho S., Silva J.C., Azuara V., Walsh M., Vallier L., Gil J. (2009). Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 18, 2134-9.

Correia, M.P., Cardoso, E.M., Pereira, C.F., Neves, R., Uhrberg, M., Arosa, F.A. (2009). Hepatocytes and IL-15: a favorable microenvironment for T cell survival and CD8+ T cell differentiation. J Immunol. 182, 6149-59.

Jorgensen, H.F., Terry, A., Beretta, C., Pereira, C.F., Leleu, M., Chen, Z.F., Kelly, C., Merkenschlager, M., Fisher, A.G. (2009). REST selectively represses a subset of RE1-containing neuronal genes in mouse embryonic stem cells. Development. 136, 715-721.

Pereira, C.F., Terranova, R., Ryan, N.K., Santos, J., Morris, K.M., Cui, W., Merkenschlager, M., Fisher, A.G. (2008). Heterokaryon-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2. PLoS Genet. 4, e1000170.

Nunes, R.J., Castro, M.A., Gonçalves, C.M., Bamberger, M., Pereira, C.F., Bismuth, G., Carmo, A.M. (2008). Protein interactions between CD2 and Lck are required for lipid raft distribution of CD2. J. Immunol. 180, 988-997.

Terranova, R., Pereira, C.F., Du Roure, C., Merkenschlager, M., and Fisher, A.G. (2006). Acquisition and extinction of gene expression programs are separable events in heterokaryon reprogramming. J. Cell Sci. 119, 2065-2072.

Cabrita, M.*, Pereira, C.F.*, Cardoso, E.M., Arosa, A. (2005). Altered expression of CD1d molecules and lipid accumulation in the human hepatoma cell line HepG2 after iron loading. FEBS J. 272, 152-165 *equal contributors.

Arosa, F.A., Pereira, C.F., Fonseca, A.M. (2004). Red blood cells as modulators of T cell growth and survival. Curr. Pharm. Des. 10, 191-201.

Fonseca, A.M., Pereira, C.F., Porto, G., Arosa, F.A. (2003). Red blood cells promote survival and cell cycle progression of human peripheral blood T cells independently of CD58/LFA-3 and heme compounds. Cell. Immunol. 224, 7-28.

Fonseca, A.M., Pereira, C.F., Porto, G., Arosa, F.A. (2003). Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress. Free Radic. Biol. Med. 35, 1404-1416.