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Programming Blood Cell Fates. Insights from Direct Lineage Conversion and Development

Capellera Garcia, Sandra LU (2018)
Abstract
Red blood cells (RBC) and platelets constitute the non-immune branch of the hematopoietic system and are responsible for the vital functions of transporting oxygen to the tissues and clotting blood vessel injuries, respectively. These cells are produced during embryonic development and throughout life through a process called hematopoiesis which is tightly regulated by extrinsic and intrinsic factors. Essential genes for RBC and/or platelet formation have been identified through targeted gene disruption strategies and studies of human diseases affecting these lineages. However, the minimal set of factors capable of initiating and specifying erythroid (RBC) and megakaryocytic (platelet) cell fate remained elusive. In this thesis, I have... (More)
Red blood cells (RBC) and platelets constitute the non-immune branch of the hematopoietic system and are responsible for the vital functions of transporting oxygen to the tissues and clotting blood vessel injuries, respectively. These cells are produced during embryonic development and throughout life through a process called hematopoiesis which is tightly regulated by extrinsic and intrinsic factors. Essential genes for RBC and/or platelet formation have been identified through targeted gene disruption strategies and studies of human diseases affecting these lineages. However, the minimal set of factors capable of initiating and specifying erythroid (RBC) and megakaryocytic (platelet) cell fate remained elusive. In this thesis, I have explored the potential of direct lineage conversion as a tool to define the master regulators of these lineages with the ultimate goal of recapitulating RBC and platelet development in vitro.
In the first paper, we employed a screen for transcription factors allowing direct induction of erythroid cell fate in mammalian fibroblasts. We identified a set of four factors (Gata1, Tal1, Lmo2 and c-Myc, or GTLM) that in eight days converted fibroblasts into induced erythroid progenitors (iEPs). iEPs exhibited properties of bona fide erythroid cells, such as morphology, gene expression, and colony-forming capacity; although their transcriptional signature resembled mainly that of primitive erythroid progenitors in the yolk sac.
In the second paper, we sought to identify missing factors and/or pathways necessary to induce adult-like erythropoiesis in fibroblasts. By comparing the transcriptome of iEPs with that of erythroid progenitors from all different layers of erythropoietic ontogeny, we identified several candidate transcription factors that are expressed in definitive erythroid progenitors, but absent in iEPs and primitive erythroid cells. These candidate genes will be tested in a future screening for modulation of developmental programming in iEPs.
In the third paper, we investigated the possibility of skewing the reprogramming process towards the megakaryocytic lineage, given that the four factors identified in the first study are implicated in the development and differentiation of the common megakaryocyte/erythroid progenitor. We found that the addition of Gata2 and Runx1 to the GTLM cocktail efficiently converted mammalian fibroblasts into megakaryocyte-like progenitors. The transdifferentiated cells expressed megakaryocytic markers, displayed polylobulated nuclei, formed megakaryocyte colonies in semisolid media, and gave rise to platelets in vitro. Moreover, transplantation of megakaryocyte-like progenitors into NSG mice resulted in engraftment and further maturation in vivo.
Overall, the results included in this thesis demonstrate that direct lineage reprogramming is a suitable tool to study erythroid and megakaryocytic cell fate regulation, and could provide a new platform to produce RBCs and platelets for personalized transfusion medicine. (Less)
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author
supervisor
opponent
  • professor Weiss, Mitchell J., Memphis
organization
publishing date
type
Thesis
publication status
published
keywords
Hematopoietic development, Erythropoiesis, Megakaryopoiesis, Direct Lineage Conversion
pages
111 pages
publisher
Lund University, Faculty of Medicine
defense location
Segerfalksalen, BMC A10, Sölvegatan 17, Lund
defense date
2018-01-16 09:00
ISBN
978-91-7619-572-7
language
English
LU publication?
yes
id
1eea6b85-7108-4be8-a0a4-e053268514bf
date added to LUP
2017-12-08 11:09:29
date last changed
2018-05-29 11:26:02
@phdthesis{1eea6b85-7108-4be8-a0a4-e053268514bf,
  abstract     = {Red blood cells (RBC) and platelets constitute the non-immune branch of the hematopoietic system and are responsible for the vital functions of transporting oxygen to the tissues and clotting blood vessel injuries, respectively. These cells are produced during embryonic development and throughout life through a process called hematopoiesis which is tightly regulated by extrinsic and intrinsic factors. Essential genes for RBC and/or platelet formation have been identified through targeted gene disruption strategies and studies of human diseases affecting these lineages. However, the minimal set of factors capable of initiating and specifying erythroid (RBC) and megakaryocytic (platelet) cell fate remained elusive. In this thesis, I have explored the potential of direct lineage conversion as a tool to define the master regulators of these lineages with the ultimate goal of recapitulating RBC and platelet development in vitro.<br/>In the first paper, we employed a screen for transcription factors allowing direct induction of erythroid cell fate in mammalian fibroblasts. We identified a set of four factors (Gata1, Tal1, Lmo2 and c-Myc, or GTLM) that in eight days converted fibroblasts into induced erythroid progenitors (iEPs). iEPs exhibited properties of bona fide erythroid cells, such as morphology, gene expression, and colony-forming capacity; although their transcriptional signature resembled mainly that of primitive erythroid progenitors in the yolk sac.<br/>In the second paper, we sought to identify missing factors and/or pathways necessary to induce adult-like erythropoiesis in fibroblasts. By comparing the transcriptome of iEPs with that of erythroid progenitors from all different layers of erythropoietic ontogeny, we identified several candidate transcription factors that are expressed in definitive erythroid progenitors, but absent in iEPs and primitive erythroid cells. These candidate genes will be tested in a future screening for modulation of developmental programming in iEPs.<br/>In the third paper, we investigated the possibility of skewing the reprogramming process towards the megakaryocytic lineage, given that the four factors identified in the first study are implicated in the development and differentiation of the common megakaryocyte/erythroid progenitor. We found that the addition of Gata2 and Runx1 to the GTLM cocktail efficiently converted mammalian fibroblasts into megakaryocyte-like progenitors. The transdifferentiated cells expressed megakaryocytic markers, displayed polylobulated nuclei, formed megakaryocyte colonies in semisolid media, and gave rise to platelets in vitro. Moreover, transplantation of megakaryocyte-like progenitors into NSG mice resulted in engraftment and further maturation in vivo.<br/>Overall, the results included in this thesis demonstrate that direct lineage reprogramming is a suitable tool to study erythroid and megakaryocytic cell fate regulation, and could provide a new platform to produce RBCs and platelets for personalized transfusion medicine.},
  author       = {Capellera Garcia, Sandra},
  isbn         = {978-91-7619-572-7},
  keyword      = {Hematopoietic development,Erythropoiesis,Megakaryopoiesis,Direct Lineage Conversion},
  language     = {eng},
  pages        = {111},
  publisher    = {Lund University, Faculty of Medicine},
  school       = {Lund University},
  title        = {Programming Blood Cell Fates. Insights from Direct Lineage Conversion and Development},
  year         = {2018},
}