This application seeks to investigate the mechanisms underlying cellular reprogramming, i.e. the conversion of adult cells into pluripotent stem cells. We will utilize transcription factor-mediated reprogramming into induced pluripotent stem cells (iPSCs) as a tool to pursue three major Aims.
In Aim 1, we will (i) test whether iPSCs derived from fibroblasts, hematopoietic and myogenic cells are transcriptionally, epigenetically and functionally distinct, (ii) study the mechanism for the increased reprogramming efficiency of progenitors, and (iii) determine if iPSC formation introduces genetic mutations into cells. Addressing the epigenetic and genetic integrity of iPSCs will be crucial for any potential therapeutic applications of this technology and may identify the most suitable cell type for generating patient-specific iPSCs.
In Aim 2, we will (i) map the transcriptional and epigenetic changes in intermediate cell populations undergoing reprogramming, (ii) test if reprogramming differentiated cells into pluripotent cells recapitulates stages of normal development, and (iii) perform a gain and loss-of-function screen, respectively, to identify novel regulators of reprogramming.
This aim will identify new molecules important during reprogramming, whose manipulation may facilitate the efficient and safe generation of patient-specific iPSCs.
In Aim 3, we will investigate the functionality of iPSCs compared with ESCs using in vitro and in vivo assays. Specifically, we will (i) assess whether neural stem cells and fibroblasts derived from iPSCs and ESCs show similar growth and differentiation characteristics in vitro, (ii) test if iPSC-derived hematopoietic stem cells are as potent as ESC-derived hematopoietic stem cells upon serial bone marrow transplantation, and (iii) produce and age entirely ESC and iPSC-derived mice to test if iPSC-derived animals age prematurely or develop cancer.
This aim will assess the safety and long-term consequences of iPSCs-derived mature cells in vivo, a prerequisite for using iPSC technology in human cell therapy.

Public Health Relevance

The goal of our lab is to dissect the mechanisms of cellular reprogramming by using induced pluripotent stem cells (iPSCs) as a tool. In this proposal, we will (i) study the role of the somatic cell-of-origin during cellular reprogramming into iPSCs, (ii) map the transcriptional and epigenetic events that occur in intermediate cells undergoing reprogramming, and (iii) compare the developmental and differentiation potentials of iPSCs and embryonic stem cells. Collectively, these experiments will give insight into the mechanisms of transcription-factor-mediated reprogramming and provide crucial information on the efficacy and safety of iPSC production for studying and potentially treating diseases.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD058013-03
Application #
8289369
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Ravindranath, Neelakanta
Project Start
2010-09-29
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$315,772
Indirect Cost
$116,572
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Bar-Nur, Ori; Brumbaugh, Justin; Verheul, Cassandra et al. (2014) Small molecules facilitate rapid and synchronous iPSC generation. Nat Methods 11:1170-6
West, Jason A; Cook, April; Alver, Burak H et al. (2014) Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming. Nat Commun 5:4719
Schwarz, Benjamin A; Bar-Nur, Ori; Silva, José C R et al. (2014) Nanog is dispensable for the generation of induced pluripotent stem cells. Curr Biol 24:347-50
Apostolou, Effie; Ferrari, Francesco; Walsh, Ryan M et al. (2013) Genome-wide chromatin interactions of the Nanog locus in pluripotency, differentiation, and reprogramming. Cell Stem Cell 12:699-712
Sarkar, Abby; Hochedlinger, Konrad (2013) The sox family of transcription factors: versatile regulators of stem and progenitor cell fate. Cell Stem Cell 12:15-30
Hirata, Akihiro; Utikal, Jochen; Yamashita, Satoshi et al. (2013) Dose-dependent roles for canonical Wnt signalling in de novo crypt formation and cell cycle properties of the colonic epithelium. Development 140:66-75
Stadtfeld, Matthias; Apostolou, Effie; Ferrari, Francesco et al. (2012) Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat Genet 44:398-405, S1-2
Wu, Sean M; Hochedlinger, Konrad (2011) Harnessing the potential of induced pluripotent stem cells for regenerative medicine. Nat Cell Biol 13:497-505
Wong, R J; Vreman, H J; Schulz, S et al. (2011) In vitro inhibition of heme oxygenase isoenzymes by metalloporphyrins. J Perinatol 31 Suppl 1:S35-41
Orkin, Stuart H; Hochedlinger, Konrad (2011) Chromatin connections to pluripotency and cellular reprogramming. Cell 145:835-50

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