Zero footprint induction of human hemogenesis to study pathologic developmental hematopoiesis in fanconi anemia
Daniel, Michael G. ; Fstkchyan, Yesai ; Gomes, Andreia ; Pereira, Filipe LU
; Lemischka, Ihor R.
and More, Kateri Ann
(2016)
In Experimental Hematology
44(9, Suppl 1).
p.65-65
- Abstract
- The inability to culture hematopoietic stem cells (HSCs) in vitro hinders the full study of physiologic and pathologic hematopoiesis “in a dish.” Recent efforts have been taken to generate HSCs via transcription factor (TF) mediated reprogramming. Most of these studies, however, rely on lentiviruses capable of genomic integration, running the risk of insertional mutagenesis and oncogenesis. Application of non-integrating self-replicating RNA (srRNA) technology can bring HSC reprogramming closer to the clinic through the generation of zero footprint hematopoietic cells. This reprogramming can also provide a novel in vitro model system to study the pathologic hematopoiesis caused by Fanconi Anemia (FA). FA associated bone marrow failure... (More)
- The inability to culture hematopoietic stem cells (HSCs) in vitro hinders the full study of physiologic and pathologic hematopoiesis “in a dish.” Recent efforts have been taken to generate HSCs via transcription factor (TF) mediated reprogramming. Most of these studies, however, rely on lentiviruses capable of genomic integration, running the risk of insertional mutagenesis and oncogenesis. Application of non-integrating self-replicating RNA (srRNA) technology can bring HSC reprogramming closer to the clinic through the generation of zero footprint hematopoietic cells. This reprogramming can also provide a novel in vitro model system to study the pathologic hematopoiesis caused by Fanconi Anemia (FA). FA associated bone marrow failure (BMF) is preceded by a significant reduction in CD34+ hematopoietic progenitors in utero, but the link between the HSC defect due to the FA mutation and this eventual BMF is incompletely understood. Using the TFs GATA2, GFI1B, and FOS (GGF), we have shown our ability to induce a hemogenic program in mouse and human fibroblasts. Polycistronic cassettes (PCs) with the GGF factors (termed PC.GGF) will be generated, inserted into srRNA backbones, and used to reprogram HDFs to obtain hematopoietic cells free of genomic integration. FA patient fibroblasts will be reprogrammed and analyzed via FACS, CFU assays, RNAseq, and ChIP-PCR to interrogate molecular processes that may be perturbed as a result of the Fanconi mutation. Our current studies identified that the TF GFI1, co-culture on the stromal layers OP9-DL1 and OP9-DL4, and transduction with PC.GGF improved the yield of triple positive CD34+CD49f+BB9+ cells, a cell surface phenotype associated with HSCs. Furthermore, OP9-DL1 co-culture imparts clonogenic potential on GGF reprogrammed HDFs. Through our ChIPseq analysis on day 2 GGF reprogrammed cells we identified a novel targeting of GATA2 to the FANC-C genomic locus. Our results demonstrate our ability to improve upon our human hemogenic reprogramming technology, and that the Fanconi pathway is activated during hemogenic induction. Through these studies we will generate the tools needed to produce hematopoietic cells free of genomic disruption, and will also apply this hemogenic induction technology to study the mechanism behind the defective hematopoiesis observed in FA. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/42478e43-d078-4208-9ae8-6e9a5064c52a
- author
- Daniel, Michael G.
; Fstkchyan, Yesai
; Gomes, Andreia
; Pereira, Filipe
LU
; Lemischka, Ihor R.
and More, Kateri Ann
- publishing date
- 2016-09-01
- type
- Contribution to journal
- publication status
- published
- in
- Experimental Hematology
- volume
- 44
- issue
- 9, Suppl 1
- pages
- 65 - 65
- publisher
- Elsevier
- ISSN
- 1873-2399
- DOI
- 10.1016/j.exphem.2016.06.112
- language
- English
- LU publication?
- no
- id
- 42478e43-d078-4208-9ae8-6e9a5064c52a
- date added to LUP
- 2023-08-10 21:35:53
- date last changed
- 2023-08-11 09:27:08
@misc{42478e43-d078-4208-9ae8-6e9a5064c52a,
abstract = {{The inability to culture hematopoietic stem cells (HSCs) in vitro hinders the full study of physiologic and pathologic hematopoiesis “in a dish.” Recent efforts have been taken to generate HSCs via transcription factor (TF) mediated reprogramming. Most of these studies, however, rely on lentiviruses capable of genomic integration, running the risk of insertional mutagenesis and oncogenesis. Application of non-integrating self-replicating RNA (srRNA) technology can bring HSC reprogramming closer to the clinic through the generation of zero footprint hematopoietic cells. This reprogramming can also provide a novel in vitro model system to study the pathologic hematopoiesis caused by Fanconi Anemia (FA). FA associated bone marrow failure (BMF) is preceded by a significant reduction in CD34+ hematopoietic progenitors in utero, but the link between the HSC defect due to the FA mutation and this eventual BMF is incompletely understood. Using the TFs GATA2, GFI1B, and FOS (GGF), we have shown our ability to induce a hemogenic program in mouse and human fibroblasts. Polycistronic cassettes (PCs) with the GGF factors (termed PC.GGF) will be generated, inserted into srRNA backbones, and used to reprogram HDFs to obtain hematopoietic cells free of genomic integration. FA patient fibroblasts will be reprogrammed and analyzed via FACS, CFU assays, RNAseq, and ChIP-PCR to interrogate molecular processes that may be perturbed as a result of the Fanconi mutation. Our current studies identified that the TF GFI1, co-culture on the stromal layers OP9-DL1 and OP9-DL4, and transduction with PC.GGF improved the yield of triple positive CD34+CD49f+BB9+ cells, a cell surface phenotype associated with HSCs. Furthermore, OP9-DL1 co-culture imparts clonogenic potential on GGF reprogrammed HDFs. Through our ChIPseq analysis on day 2 GGF reprogrammed cells we identified a novel targeting of GATA2 to the FANC-C genomic locus. Our results demonstrate our ability to improve upon our human hemogenic reprogramming technology, and that the Fanconi pathway is activated during hemogenic induction. Through these studies we will generate the tools needed to produce hematopoietic cells free of genomic disruption, and will also apply this hemogenic induction technology to study the mechanism behind the defective hematopoiesis observed in FA.}},
author = {{Daniel, Michael G. and Fstkchyan, Yesai and Gomes, Andreia and Pereira, Filipe and Lemischka, Ihor R. and More, Kateri Ann}},
issn = {{1873-2399}},
language = {{eng}},
month = {{09}},
note = {{Conference Abstract}},
number = {{9, Suppl 1}},
pages = {{65--65}},
publisher = {{Elsevier}},
series = {{Experimental Hematology}},
title = {{Zero footprint induction of human hemogenesis to study pathologic developmental hematopoiesis in fanconi anemia}},
url = {{http://dx.doi.org/10.1016/j.exphem.2016.06.112}},
doi = {{10.1016/j.exphem.2016.06.112}},
volume = {{44}},
year = {{2016}},
}