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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.


Related in: MedlinePlus

SLRB-iHP cells engraft for up to 4 months in vivo.(a) Representative FACS plot of SLRB/SLRH-iHP (tdTomato+) cells in PB and SP at 16 wpt (Wpt: week post-transplant). SLR factors were delivered in one polycistronic construct. 5 × 106 iHP cells were transplanted per mouse. Mice analysed: SLRB iHP (n=9), SLRH-HP (n=4). These are representative of three independent experiments. (b) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in PB at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP, NOD-SCID (NS) mice n=6 (5 wpt) and n=9 (16 wpt) were analysed. For SLRH-iHP, different mouse strains were analysed. At 5wpt, NS mice n=5, NSG mice n=3, C57BL/6 (B6) mice n=3. At 16 wpt, NS mice n=3, NSG mice n=1, B6 mice n=2. Only tdTomato+ cell contribution ≥0.1% are plotted. Data drawn from three independent experiments. (c) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in SP at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP cells, mice n=5 at 5wpt, and n=9 at 16 wpt. For SLRH-iHP cells, mice analysed: n=5 at 5wpt, n=4 at 16 wpt. Only tdTomato+ cells contribution ≥0.1% were plotted. These data are from three independent experiments. (d) Representative FACS plot of multilineage reconstitution of SLRB/H-iHP (tdTomato+) cells in SP at 16 wpt. SLR factors were delivered in one polycistronic construct. Percentage of cells are shown as mean±s.d. (for SLRB, n=6 mice; for SLRH, n=3 mice). These data are representative of three independent experiments. (e) SLRB-iHP (tdTomato+) cells in PB at 16 wpt. SLR factors were delivered in one polycistronic construct. tdTomato− PB cells from respective CD45.1+ recipient mice shown as control (Ctr). These data are representative of three independent experiments.
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f3: SLRB-iHP cells engraft for up to 4 months in vivo.(a) Representative FACS plot of SLRB/SLRH-iHP (tdTomato+) cells in PB and SP at 16 wpt (Wpt: week post-transplant). SLR factors were delivered in one polycistronic construct. 5 × 106 iHP cells were transplanted per mouse. Mice analysed: SLRB iHP (n=9), SLRH-HP (n=4). These are representative of three independent experiments. (b) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in PB at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP, NOD-SCID (NS) mice n=6 (5 wpt) and n=9 (16 wpt) were analysed. For SLRH-iHP, different mouse strains were analysed. At 5wpt, NS mice n=5, NSG mice n=3, C57BL/6 (B6) mice n=3. At 16 wpt, NS mice n=3, NSG mice n=1, B6 mice n=2. Only tdTomato+ cell contribution ≥0.1% are plotted. Data drawn from three independent experiments. (c) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in SP at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP cells, mice n=5 at 5wpt, and n=9 at 16 wpt. For SLRH-iHP cells, mice analysed: n=5 at 5wpt, n=4 at 16 wpt. Only tdTomato+ cells contribution ≥0.1% were plotted. These data are from three independent experiments. (d) Representative FACS plot of multilineage reconstitution of SLRB/H-iHP (tdTomato+) cells in SP at 16 wpt. SLR factors were delivered in one polycistronic construct. Percentage of cells are shown as mean±s.d. (for SLRB, n=6 mice; for SLRH, n=3 mice). These data are representative of three independent experiments. (e) SLRB-iHP (tdTomato+) cells in PB at 16 wpt. SLR factors were delivered in one polycistronic construct. tdTomato− PB cells from respective CD45.1+ recipient mice shown as control (Ctr). These data are representative of three independent experiments.

Mentions: Upon transplantation into irradiated NOD-SCID (NS) mice, both SLRH-iHP and SLRB-iHP (in which transgenes were constitutively expressed) contributed to all hematopoietic organs—PB (Fig. 3a,b, Supplementary Fig. 4a), SP (Fig. 3a, c, Supplementary Fig. 4a) and BM (Supplementary Figs. 4a and 5a–c)—for up to 16 weeks. SLRB-iHPs were significantly more robust at PB reconstitution at 5 weeks post-transplant (wpt) and 16 wpt by comparison with SLRH-iHP (Fig. 3a,b, Supplementary Fig. 4a). In contrast, SLRH-iHP showed higher BM engraftment at 16 wpt than SLRB-iHP (Supplementary Fig. 5a–c).


Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors
SLRB-iHP cells engraft for up to 4 months in vivo.(a) Representative FACS plot of SLRB/SLRH-iHP (tdTomato+) cells in PB and SP at 16 wpt (Wpt: week post-transplant). SLR factors were delivered in one polycistronic construct. 5 × 106 iHP cells were transplanted per mouse. Mice analysed: SLRB iHP (n=9), SLRH-HP (n=4). These are representative of three independent experiments. (b) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in PB at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP, NOD-SCID (NS) mice n=6 (5 wpt) and n=9 (16 wpt) were analysed. For SLRH-iHP, different mouse strains were analysed. At 5wpt, NS mice n=5, NSG mice n=3, C57BL/6 (B6) mice n=3. At 16 wpt, NS mice n=3, NSG mice n=1, B6 mice n=2. Only tdTomato+ cell contribution ≥0.1% are plotted. Data drawn from three independent experiments. (c) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in SP at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP cells, mice n=5 at 5wpt, and n=9 at 16 wpt. For SLRH-iHP cells, mice analysed: n=5 at 5wpt, n=4 at 16 wpt. Only tdTomato+ cells contribution ≥0.1% were plotted. These data are from three independent experiments. (d) Representative FACS plot of multilineage reconstitution of SLRB/H-iHP (tdTomato+) cells in SP at 16 wpt. SLR factors were delivered in one polycistronic construct. Percentage of cells are shown as mean±s.d. (for SLRB, n=6 mice; for SLRH, n=3 mice). These data are representative of three independent experiments. (e) SLRB-iHP (tdTomato+) cells in PB at 16 wpt. SLR factors were delivered in one polycistronic construct. tdTomato− PB cells from respective CD45.1+ recipient mice shown as control (Ctr). These data are representative of three independent experiments.
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Related In: Results  -  Collection

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f3: SLRB-iHP cells engraft for up to 4 months in vivo.(a) Representative FACS plot of SLRB/SLRH-iHP (tdTomato+) cells in PB and SP at 16 wpt (Wpt: week post-transplant). SLR factors were delivered in one polycistronic construct. 5 × 106 iHP cells were transplanted per mouse. Mice analysed: SLRB iHP (n=9), SLRH-HP (n=4). These are representative of three independent experiments. (b) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in PB at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP, NOD-SCID (NS) mice n=6 (5 wpt) and n=9 (16 wpt) were analysed. For SLRH-iHP, different mouse strains were analysed. At 5wpt, NS mice n=5, NSG mice n=3, C57BL/6 (B6) mice n=3. At 16 wpt, NS mice n=3, NSG mice n=1, B6 mice n=2. Only tdTomato+ cell contribution ≥0.1% are plotted. Data drawn from three independent experiments. (c) Summary of SLRB/SLRH-iHP (tdTomato+) cell engraftment in SP at 5 and 16 wpt. SLR factors were delivered in one polycistronic construct. For SLRB-iHP cells, mice n=5 at 5wpt, and n=9 at 16 wpt. For SLRH-iHP cells, mice analysed: n=5 at 5wpt, n=4 at 16 wpt. Only tdTomato+ cells contribution ≥0.1% were plotted. These data are from three independent experiments. (d) Representative FACS plot of multilineage reconstitution of SLRB/H-iHP (tdTomato+) cells in SP at 16 wpt. SLR factors were delivered in one polycistronic construct. Percentage of cells are shown as mean±s.d. (for SLRB, n=6 mice; for SLRH, n=3 mice). These data are representative of three independent experiments. (e) SLRB-iHP (tdTomato+) cells in PB at 16 wpt. SLR factors were delivered in one polycistronic construct. tdTomato− PB cells from respective CD45.1+ recipient mice shown as control (Ctr). These data are representative of three independent experiments.
Mentions: Upon transplantation into irradiated NOD-SCID (NS) mice, both SLRH-iHP and SLRB-iHP (in which transgenes were constitutively expressed) contributed to all hematopoietic organs—PB (Fig. 3a,b, Supplementary Fig. 4a), SP (Fig. 3a, c, Supplementary Fig. 4a) and BM (Supplementary Figs. 4a and 5a–c)—for up to 16 weeks. SLRB-iHPs were significantly more robust at PB reconstitution at 5 weeks post-transplant (wpt) and 16 wpt by comparison with SLRH-iHP (Fig. 3a,b, Supplementary Fig. 4a). In contrast, SLRH-iHP showed higher BM engraftment at 16 wpt than SLRB-iHP (Supplementary Fig. 5a–c).

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.


Related in: MedlinePlus