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Cell reprogramming and developmental hematopoiesis

Research Support

Title: "Mechanisms underlying hemogenic induction in human fibroblasts"

Reference: PTDC/BIM-MED/0075/2014 (POCI-01-0145-FEDER-016689)

Amounts involved:

Total Investment: 199.687,00€

CNC: 199.687,00€

FEDER funding support (85%): 169.733,95€

OE funding support: 29.953,05€

Resume:
Hematopoietic Stem Cell (HSC) transplantation whether from bone marrow, mobilized peripheral blood or cord blood
is the best example of regenerative cell therapy. HSCs have remarkable therapeutic applications in a broad range
of diseases that affect the blood forming and immune system. Nevertheless, there remains a shortage of usable
genetically-matched material for all needs. Growing HSCs in vitro from adult or fetal sources or generating them
from embryonic stem cells has proven to be challenging despite of 20 years of intensive research. An alternative
approach would be to generate transplantable HSCs directly from patients? own adult cells using programming
technology. Recently we demonstrated direct reprogramming of mouse fibroblasts into clonogenic hematopoietic
progenitors with the combination of transcription factors (TFs), Gata2, Gfi1b, cFos and Etv6. These four TFs induce
a dynamic, multi-stage hemogenic process that progresses through an endothelial-like intermediate. As such, it
appears to recapitulate developmental hematopoiesis in vitro. We now have strong evidence that the same blood
forming process can be induced in human skin-derived fibroblasts with GATA2, GFI1B, and cFOS. The reprogrammed
fibroblasts undergo morphological and transcriptional changes, display cell surface phenotypes of human HSC,
and repopulate immunodefiecient NSG mice. We first propose to characterize the human induction process using
cell surface phenotypes, global gene expression analyses and functional assays. We further propose to dissect
the molecular mechanisms of such induction by the identified group of TFs. Specifically, we will investigate the
mechanisms that mediate this process by studying how these factors modify the epigenome and therefore directing
and establishing a new cell identity. We propose to map TF binding, DNA methylation and histone modifications at a
genome-wide level during the induction process. The dissection of such molecular mechanisms may ultimately allow
for the use of programmed cells in the clinic. The successful generation of directly programmed HSCs will have a
highly significant impact not only on patient specific therapeutics for immuno-hematopoietic diseases but also provide
novel technologies to correct genetic disorders.

Collaborations:

Caetano Reis e Sousa Francis, Crick Institute, London; UK

Marella de Bruijn, Weatherall Institute of Molecular Medicine, Oxford; UK

Kateri Moore, Icahn School of Medicine at Mount Sinai, New York, USA

Publicizing Sheet

....................................................................................................................................................................................

Title: “ReproDC .: Translação da reprogramação celular direta em células dendríticas na imunoterapia do cancro.”

Referência: CENTRO-01-0145-FEDER-039473 “ReproDC .: Translação da reprogramação celular direta em células dendríticas na imunoterapia do cancro.”

Amounts involved:

Investimento Total: 48.925,50€

CNC funding support: 48.925,50 €

FEDER funding support : (85%): 41586,68€

Resume:
This project reffers to a PI protection request for the technology COMPOSITIONS FOR REPROGRAMMING CELLS INTO DENDRITIC CELLS OR ANTIGEN PRESENTING CELLS-METHODS AND USES THEREOF, which has the potential for application in the development of cancer immunotherapies based in celular reprogramming.

Carlos F. Pereira

Group Leader

Principal Investigator

Cristiana Pires

Post-Doctoral Fellow

Fábio Rosa

PhD student

Alexandra Ferreira

PhD student

Marta Laranjeiro Pinto

Post-Doctoral Fellow

Catarina Silva

Master Student

Rita Alves

PhD student

Inês Caiado

MSc student

Susana Pedreiro

Technician/ Lab Manager

Luis Oliveira

MSc student

Highly motivated undergraduates, PhD students and Postdocs interested in joining the Cell Reprogramming laboratory are encouraged to contact the PI – please send an email with your CV attached.

Alumni

Selected Publications

Rosa, F.F. *, Pires, C.F. *, Kurochkin, I., Ferreira, A.G., Gomes, A.M., Palma, L.G., Shaiv, K., Solanas, L., Azenha, C., Papatsenko, D., Schulx, O., Reis e Sousa, C., Pereira, C.F. (2018) Direct reprogramming of fibroblasts into antigen-presenting dendritic cells. Science Immunology 7, 3(30). *equal contribution. -Cover Science Immunology issue December 2018. Preview by Yona & Mildner, issue December 2018.

Gomes, A.M., Kurochkin, I., Chang, B.; Daniel, M., Law, K., Satija, N., Lachmann, A., Wang, Z., Ferreira, L., Ma’ayan, A., Chen, B.K., Papatsenko, D., Lemischka, I.R., Moore, K.A., Pereira, C.F. (2018) Cooperative Transcription Factor Induction Mediates Hemogenic Reprogramming. Cell Reports 25, 2821-25.

Kirkeby, A., Perlmann, T., Pereira, C. F *. (2016) The stem cell niche finds its true north. Development. *corresponding author.

Pereira, C.F.*; Chang, B.; Papatsenko, D.; Gomes, A.; Bernitz, B.; Niu, X.; Swiers, G.; Azzoni, E.; Brujin M.F.T.R.; Schaniel, C.; Lemischka, I.R.; Moore, K.A. (2016) Identification of Hemogenic Precursors in Placenta that Give Rise to Definitive Hematopoietic Stem Cells In Vivo is Informed by Hematopoietic Reprogramming In Vitro. Developmental Cell 36, 525-39. *corresponding author.
- Preview by Calvanese & Mikkola, 2016.

Pereira, C.F. *, Chang, B., Qiu, J., Niu, X., Papatsenko D., Hendry, C.E., Clark, N.R., Nomura-Kitabayashi, A., Kovacic, J., Ma’ayan A., Schaniel C., Lemischka, I.R., Moore, K.A. (2013) Induction of a Hemogenic Program in Mouse Fibroblasts. Cell Stem Cell 13, 205-18. *corresponding author. -Cover Cell Stem Cell issue August 2013. Preview by Pfaff & Cantz, 2013. -Highlighted in Molecular Therapy 21, 1291 (2013), Stem cells Translational Medicine News section June 2013. -Recommended by the Faculty of 1000.

Pereira C.F. *, Lemischka I.R., Moore K.A. (2012) Reprogramming cell fates: insights from combinatorial approaches. Ann. N. Y. Acad. Sci. 1266, 7-17. *corresponding author.

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

See more

Daniel, M. G., Pereira, C. F., Lemischka, I. R., Moore, K. A. (2015) Making a Hematopoietic Stem Cell. Trends in cell biology 26:212-14.

Waghray, A., Saiz, N., Jayaprakash A.D., Freire, A., Papatsenko, D., Pereira, C.F., Lee, D.F., Brosh, R., Chang, B., Darr, H., Gingold, J., Kelley, K., Schaniel, C., Hadjantonakis, A.K., Lemischka, I.R. (2015) Tbx3 Controls Dppa3 Levels and Exit from Pluripotency toward Mesoderm. Stem Cell Reports. 15, 00152-6.

Pereira C.F. *, Lemischka I.R., Moore K.A*. (2014) “From Blood to Blood” de-differentiation of hematopoietic progenitors to stem cells. EMBO J. 14, 1511-3. *corresponding author.

Fidalgo M., Faiola F., Pereira C.F., Ding J., Saunders A., Gingold J., Schaniel C., Lemischka I.R., Silva J.C., Wang J. (2012) Zfp281 mediates Nanog autorepression through recruitment of the NuRD complex and inhibits somatic cell reprogramming. PNAS 109, 16202-7.

Lee D.F., Su J., Ang Y.S., Carvajal-Vergara X., Mulero-Navarro S., Pereira C.F., Gingold J., Wang H.L., Zhao R., Sevilla A., Darr H., et al. (2012). Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling. Cell Stem Cell. 11, 179-194. - Highlighted in Nature Cell Biology 14, 990 (2012).

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.

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.

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.

Patents

Rosa, F.F, Pires, C.F., Pereira, C.F. Compositions for reprogramming cells into dendritic cells or antigen presenting cells, methods and uses thereof. WO/2018/185709.

Pereira, C.F., Moore, K.A., Lemischka, I.R. Method for programming differentiated cells into hematopoietic stem cells. US-2015-0004145-A1.

Covers

On the cover: "Induced" Dendritic Cells. In this issue, Rosa et al. report that ectopic expression of three transcription factors—PU.1, IRF8, and BATF3—is sufficient to reprogram mouse and human fibroblasts into "induced" dendritic cells. Featured on the cover is a colored scanning electron micrograph showing an "induced" dendritic cell with prominent cytoplasmic protrusions. CREDIT: JONAS AHLSTEDT & SEBASTIAN WASSERSTROM

On the cover: In this issue, Pereira et al. (205–218) describe the induction of hemogenesis in mouse fibroblasts via transduction of the transcription factors Gata2, cFos, Gfi1b, and Etv6. The cover is an artistic representation of this induction process. In a futuristic setting, a scientist programs a computer to program blood, leading to the formation of hemogenic blood vessels and subsequently blood. Cover design and illustration by Carlos-Filipe Pereira and Sandeep Menon.

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

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

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.

GROUP LEADER

Carlos Filipe Pereira, Ph.D.
Assistant Investigator (equiv. Assistant Professor)
Tel: +351 231 249 170
Fax: +351 231 249 179

Address:
CNC/UC-Biotech
Parque Tecnológico de Cantanhede,
Núcleo 4, Lote 8
3060-197 - Cantanhede
PORTUGAL

2016: FCT Investigator Programme, CNC, University of Coimbra, Portugal.
2015-2017: Marie Curie International Incoming Fellow, University of Coimbra, Portugal.
2013-2015: Revson Foundation Senior post-doctoral research fellow in Biomedical Science, Icahn School of Medicine at Mount Sinai, New York, USA.
2010-2012: EMBO post-doctoral research fellow, Icahn School of Medicine at Mount Sinai, New York, USA.
2009-2010: Post-doctoral fellow, Imperial College, London, UK.
2009: Ph.D., Imperial College, London, UK.
2003-2008: Graduate Program in Areas of Basic and Applied Biology (GABBA), University of Porto, Portugal.
2002: BSc in Biology, University of Porto, Portugal.

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Funded by ERDF - European Regional Development Fund through COMPETE 2020 and National Funds via FCT - Fundação para a Ciência e Tecnologia, under projects POCI-01-0145-FEDER-007440 e UIDB/04539/2020.

Instituto de Investigação Interdisciplinar Universidade de Coimbra

Centre for Innovative Biomedicine and Biotechnology