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Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

View Article: PubMed Central - PubMed

ABSTRACT

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.

No MeSH data available.


SLRB-iHPs generate B cells in vivo.V(D)J recombination events and transgene integration were detected in SLRB-iHP-derived B cells (iHP-B cells) at single-cell level. iHP-B cells are from 16 wpt spleens. Numbers at top of the panel are cell ID of individual single cells. Con (B6) denotes control (Con) cells from the SP of C57BL/6 (B6) mice. MEF (14 dpi) are cells from SLRB infected tdTomato+ MEF at 14 dpi. Sp (B6) and Sp (tdTomato+) stand for total spleen cells. VH7183-DJH and DHQ52-JH are used to reveal V to DJ and D to J recombination events at heavy chain, respectively. Vk−Jk denotes V to J recombination at Kappa chain. WT (Wild type) and MT (tdTomato mutant) show the genotype of the cells. SLR-T and B-T stand for transgene integration of SLR (Polycistronic) and Bmi1. Polycistronic SLR was used for the generation of iHP-B cells and 14 dpi MEF. These are representative of two independent experiments.
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f4: SLRB-iHPs generate B cells in vivo.V(D)J recombination events and transgene integration were detected in SLRB-iHP-derived B cells (iHP-B cells) at single-cell level. iHP-B cells are from 16 wpt spleens. Numbers at top of the panel are cell ID of individual single cells. Con (B6) denotes control (Con) cells from the SP of C57BL/6 (B6) mice. MEF (14 dpi) are cells from SLRB infected tdTomato+ MEF at 14 dpi. Sp (B6) and Sp (tdTomato+) stand for total spleen cells. VH7183-DJH and DHQ52-JH are used to reveal V to DJ and D to J recombination events at heavy chain, respectively. Vk−Jk denotes V to J recombination at Kappa chain. WT (Wild type) and MT (tdTomato mutant) show the genotype of the cells. SLR-T and B-T stand for transgene integration of SLR (Polycistronic) and Bmi1. Polycistronic SLR was used for the generation of iHP-B cells and 14 dpi MEF. These are representative of two independent experiments.

Mentions: To further test the authenticity of iHP-derived B-lymphoid cells (wild type for p53), we assessed at their ability to undergo DNA recombination at the IgH and IgL gene loci (known as V(D)J recombination) at the 16 wpt timepoint. Recombination events were detected in iHP-derived B cells at either the heavy chain or kappa chain genes at single-cell level (Fig. 4, Supplementary Fig. 10). When genotyped, all the donor single cells were homozygous for the tdTomato knock-in and integrations of both SLR (polycistronic) and Bmi1 transgenes were detected in the genome, confirming that they were indeed derived from iHP cells (Fig. 4, Supplementary Fig. 10).


Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors
SLRB-iHPs generate B cells in vivo.V(D)J recombination events and transgene integration were detected in SLRB-iHP-derived B cells (iHP-B cells) at single-cell level. iHP-B cells are from 16 wpt spleens. Numbers at top of the panel are cell ID of individual single cells. Con (B6) denotes control (Con) cells from the SP of C57BL/6 (B6) mice. MEF (14 dpi) are cells from SLRB infected tdTomato+ MEF at 14 dpi. Sp (B6) and Sp (tdTomato+) stand for total spleen cells. VH7183-DJH and DHQ52-JH are used to reveal V to DJ and D to J recombination events at heavy chain, respectively. Vk−Jk denotes V to J recombination at Kappa chain. WT (Wild type) and MT (tdTomato mutant) show the genotype of the cells. SLR-T and B-T stand for transgene integration of SLR (Polycistronic) and Bmi1. Polycistronic SLR was used for the generation of iHP-B cells and 14 dpi MEF. These are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121332&req=5

f4: SLRB-iHPs generate B cells in vivo.V(D)J recombination events and transgene integration were detected in SLRB-iHP-derived B cells (iHP-B cells) at single-cell level. iHP-B cells are from 16 wpt spleens. Numbers at top of the panel are cell ID of individual single cells. Con (B6) denotes control (Con) cells from the SP of C57BL/6 (B6) mice. MEF (14 dpi) are cells from SLRB infected tdTomato+ MEF at 14 dpi. Sp (B6) and Sp (tdTomato+) stand for total spleen cells. VH7183-DJH and DHQ52-JH are used to reveal V to DJ and D to J recombination events at heavy chain, respectively. Vk−Jk denotes V to J recombination at Kappa chain. WT (Wild type) and MT (tdTomato mutant) show the genotype of the cells. SLR-T and B-T stand for transgene integration of SLR (Polycistronic) and Bmi1. Polycistronic SLR was used for the generation of iHP-B cells and 14 dpi MEF. These are representative of two independent experiments.
Mentions: To further test the authenticity of iHP-derived B-lymphoid cells (wild type for p53), we assessed at their ability to undergo DNA recombination at the IgH and IgL gene loci (known as V(D)J recombination) at the 16 wpt timepoint. Recombination events were detected in iHP-derived B cells at either the heavy chain or kappa chain genes at single-cell level (Fig. 4, Supplementary Fig. 10). When genotyped, all the donor single cells were homozygous for the tdTomato knock-in and integrations of both SLR (polycistronic) and Bmi1 transgenes were detected in the genome, confirming that they were indeed derived from iHP cells (Fig. 4, Supplementary Fig. 10).

View Article: PubMed Central - PubMed

ABSTRACT

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.

No MeSH data available.