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- Capellera-Garcia, Sandra, et al.
(författare)
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Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion
- 2016
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 15:11, s. 2550-2562
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Tidskriftsartikel (refereegranskat)abstract
- Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.
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| 2. |
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| 3. |
- Capellera Garcia, Sandra
(författare)
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Programming Blood Cell Fates. Insights from Direct Lineage Conversion and Development
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.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.
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| 4. |
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| 5. |
- Ilsley, Melissa, et al.
(författare)
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Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors
- 2018
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Ingår i: Journal of visualized experiments : JoVE. - : MyJove Corporation. - 1940-087X. ; :142
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Tidskriftsartikel (refereegranskat)abstract
- Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.
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| 6. |
- Pulecio, Julian, et al.
(författare)
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Direct Conversion of Fibroblasts to Megakaryocyte Progenitors
- 2016
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 17:3, s. 671-683
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Tidskriftsartikel (refereegranskat)abstract
- Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41(+), display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.
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