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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.


Transcriptional dynamics and Lmo2 genomic binding during iHP reprogramming.(a) Schema of the transdifferentiation procedure with days indicating when samples were collected for microarray and ChIP-Seq analyses. (b) Hierarchical clustering of D0, D4, iHP (Kit+ and CD45+ cells) and BM (Kit+ and CD45+ cells) with published HSPC microarray datasets40. Red boxes denote the samples generated in this study whereas the other samples are HSPCs from published datasets40. In brief, HSPCs from published datasets are VE-cadherin+CD45+ cells in AGM, Lin−Sca1+Kit+VE-cadherin+Mac-1low cells in E12.5 foetal liver, CD45+Kit+CD34mid cells in E12.5 placenta, Lin−Sca1+Kit+CD150+CD48− cells in E13.5 and E14.5 foetal liver, CD41+Kit+CD34+ cells in yolk sac, Kit+CD41+ cells in day 6 embryoid bodies (EB), and CD41brightCD45−CD34− ESC-derived HSC-like cells. (c) Dynamic gene expression during the microarray timecourse, as assayed by GEDI. The GEDI plots demonstrate a transition state in 14 dpi cells and a major change of the gene expression profile at 26 dpi cells during reprogramming. (d) Motif analysis for Lmo2-bound peaks at 4 dpi shows an enrichment of motifs recognized by other hematopoietic transcription factors. (e) Clustered heatmaps of MEF H3K4me3, H3K27ac and H3K27me3 signals at genomic regions bound by Lmo2 at 4 dpi. The heatmaps indicate that 4 dpi Lmo2 binds both closed and open chromatin. (f) GO analysis of Lmo2-bound genes; bar plots indicate P values of the overrepresented pathways and biological processes.
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f5: Transcriptional dynamics and Lmo2 genomic binding during iHP reprogramming.(a) Schema of the transdifferentiation procedure with days indicating when samples were collected for microarray and ChIP-Seq analyses. (b) Hierarchical clustering of D0, D4, iHP (Kit+ and CD45+ cells) and BM (Kit+ and CD45+ cells) with published HSPC microarray datasets40. Red boxes denote the samples generated in this study whereas the other samples are HSPCs from published datasets40. In brief, HSPCs from published datasets are VE-cadherin+CD45+ cells in AGM, Lin−Sca1+Kit+VE-cadherin+Mac-1low cells in E12.5 foetal liver, CD45+Kit+CD34mid cells in E12.5 placenta, Lin−Sca1+Kit+CD150+CD48− cells in E13.5 and E14.5 foetal liver, CD41+Kit+CD34+ cells in yolk sac, Kit+CD41+ cells in day 6 embryoid bodies (EB), and CD41brightCD45−CD34− ESC-derived HSC-like cells. (c) Dynamic gene expression during the microarray timecourse, as assayed by GEDI. The GEDI plots demonstrate a transition state in 14 dpi cells and a major change of the gene expression profile at 26 dpi cells during reprogramming. (d) Motif analysis for Lmo2-bound peaks at 4 dpi shows an enrichment of motifs recognized by other hematopoietic transcription factors. (e) Clustered heatmaps of MEF H3K4me3, H3K27ac and H3K27me3 signals at genomic regions bound by Lmo2 at 4 dpi. The heatmaps indicate that 4 dpi Lmo2 binds both closed and open chromatin. (f) GO analysis of Lmo2-bound genes; bar plots indicate P values of the overrepresented pathways and biological processes.

Mentions: To evaluate the ordered series of molecular events driving fibroblasts to a hematopoietic fate, we performed microarray transcriptome analysis of tdTomato+ fibroblasts (MEFs), intermediate populations undergoing SLHR reprogramming (4 dpi and 14 dpi), reprogrammed 26 dpi iHPs (CD45+ or Kit+) and finally BM (CD45+ or Kit+) cells as a positive control (Fig. 5a). We also profiled the genome-wide binding of transcription factor Lmo2 at an incipient stage of SLHR reprogramming (namely, 4 dpi; Fig. 5a).


Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors
Transcriptional dynamics and Lmo2 genomic binding during iHP reprogramming.(a) Schema of the transdifferentiation procedure with days indicating when samples were collected for microarray and ChIP-Seq analyses. (b) Hierarchical clustering of D0, D4, iHP (Kit+ and CD45+ cells) and BM (Kit+ and CD45+ cells) with published HSPC microarray datasets40. Red boxes denote the samples generated in this study whereas the other samples are HSPCs from published datasets40. In brief, HSPCs from published datasets are VE-cadherin+CD45+ cells in AGM, Lin−Sca1+Kit+VE-cadherin+Mac-1low cells in E12.5 foetal liver, CD45+Kit+CD34mid cells in E12.5 placenta, Lin−Sca1+Kit+CD150+CD48− cells in E13.5 and E14.5 foetal liver, CD41+Kit+CD34+ cells in yolk sac, Kit+CD41+ cells in day 6 embryoid bodies (EB), and CD41brightCD45−CD34− ESC-derived HSC-like cells. (c) Dynamic gene expression during the microarray timecourse, as assayed by GEDI. The GEDI plots demonstrate a transition state in 14 dpi cells and a major change of the gene expression profile at 26 dpi cells during reprogramming. (d) Motif analysis for Lmo2-bound peaks at 4 dpi shows an enrichment of motifs recognized by other hematopoietic transcription factors. (e) Clustered heatmaps of MEF H3K4me3, H3K27ac and H3K27me3 signals at genomic regions bound by Lmo2 at 4 dpi. The heatmaps indicate that 4 dpi Lmo2 binds both closed and open chromatin. (f) GO analysis of Lmo2-bound genes; bar plots indicate P values of the overrepresented pathways and biological processes.
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Related In: Results  -  Collection

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f5: Transcriptional dynamics and Lmo2 genomic binding during iHP reprogramming.(a) Schema of the transdifferentiation procedure with days indicating when samples were collected for microarray and ChIP-Seq analyses. (b) Hierarchical clustering of D0, D4, iHP (Kit+ and CD45+ cells) and BM (Kit+ and CD45+ cells) with published HSPC microarray datasets40. Red boxes denote the samples generated in this study whereas the other samples are HSPCs from published datasets40. In brief, HSPCs from published datasets are VE-cadherin+CD45+ cells in AGM, Lin−Sca1+Kit+VE-cadherin+Mac-1low cells in E12.5 foetal liver, CD45+Kit+CD34mid cells in E12.5 placenta, Lin−Sca1+Kit+CD150+CD48− cells in E13.5 and E14.5 foetal liver, CD41+Kit+CD34+ cells in yolk sac, Kit+CD41+ cells in day 6 embryoid bodies (EB), and CD41brightCD45−CD34− ESC-derived HSC-like cells. (c) Dynamic gene expression during the microarray timecourse, as assayed by GEDI. The GEDI plots demonstrate a transition state in 14 dpi cells and a major change of the gene expression profile at 26 dpi cells during reprogramming. (d) Motif analysis for Lmo2-bound peaks at 4 dpi shows an enrichment of motifs recognized by other hematopoietic transcription factors. (e) Clustered heatmaps of MEF H3K4me3, H3K27ac and H3K27me3 signals at genomic regions bound by Lmo2 at 4 dpi. The heatmaps indicate that 4 dpi Lmo2 binds both closed and open chromatin. (f) GO analysis of Lmo2-bound genes; bar plots indicate P values of the overrepresented pathways and biological processes.
Mentions: To evaluate the ordered series of molecular events driving fibroblasts to a hematopoietic fate, we performed microarray transcriptome analysis of tdTomato+ fibroblasts (MEFs), intermediate populations undergoing SLHR reprogramming (4 dpi and 14 dpi), reprogrammed 26 dpi iHPs (CD45+ or Kit+) and finally BM (CD45+ or Kit+) cells as a positive control (Fig. 5a). We also profiled the genome-wide binding of transcription factor Lmo2 at an incipient stage of SLHR reprogramming (namely, 4 dpi; Fig. 5a).

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.