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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.

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Runx1 together with HoxB4 or Bmi1 augments the CFU-S forming-ability of iHP cells.(a) Similar to control bone marrow cells (BM Ctr, tdTomato+), SLHR-iHP cells (tdTomato+) form tdTomato+ nodules in the spleen at 12 days post-transplantation (dpt) into lethally irradiated C57Bl/6 mice (CFU-S12). Factors were singly delivered in individual pMX constructs. 1 × 105 BM control cells, 5 × 106 SLHR-iHP cells or 1 × 106 Kit+ cells were transplanted per mouse. Mice analysed: BM Ctr, n=4, SLHR-iHP cells, n=20, SLHR-iHP kit+ cells, n=4. These are representative of three independent experiments. Scale bar, 2 mm. (b) Comparison of CFU-S12 potential of iHP cells induced by differing TF cocktails. SL-, SLB-, SLHR-, SLRH- or SLRB-iHP cells (tdTomato+) were transplanted into lethally irradiated SCID mice. Factors were delivered in pMXconstructs; SLHR denotes individual delivery of S, L, H and R; SLRH and SLRB denotes use of a polycistronic construct containing S, L and R in one pMX vector, together with individual delivery of either H or B in a separate construct. For SL-, SLB-, SLHR-iHP, 5 × 106 cells were transplanted. For SLRH- and SLRB-iHPs, 2 × 106 cells were transplanted. Mice analysed: for SL-, SLB-, SLHR-iHP, n=6, for SLRB- and SLRH-iHP, n=12 each. Scale bar: 2 mm. These are representative of three independent experiments. (c) Frequency of CFU-S12 of different iHP cells. iHP cells are named as in b. Data are shown as mean±s.d. per spleen. These data are from three independent experiments. (d) Representative FACS analysis of tdTomato+ cells in BM of SCID mice at 12–14 dpt. SLR factors were delivered in one polycistronic construct. For SLRB/H-iHP cells: 2 × 106 cells per mouse were transplanted; for Ctr BM cells (tdTomato+), 2 × 105 cells per mouse were transplanted. Percentage of tdTomato+ cells are shown as mean±s.d. (n=6 mice for each type of cells), representative from three independent experiments. (e) Representative FACS analysis of tdTomato+ cells stained with lineage markers (shown on the plots) in the BM of SCID mice transplanted with either SLRB/SLRH-HP cells and control BM cells (Ctr tdTomato+) at 12–14dpt. SLR factors were delivered in one polycistronic construct. Mice analysed: n=6 for each type of cells. Ery, Erythroid; Meg, Megakaryocytes. These data are representative of three independent experiments.
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f2: Runx1 together with HoxB4 or Bmi1 augments the CFU-S forming-ability of iHP cells.(a) Similar to control bone marrow cells (BM Ctr, tdTomato+), SLHR-iHP cells (tdTomato+) form tdTomato+ nodules in the spleen at 12 days post-transplantation (dpt) into lethally irradiated C57Bl/6 mice (CFU-S12). Factors were singly delivered in individual pMX constructs. 1 × 105 BM control cells, 5 × 106 SLHR-iHP cells or 1 × 106 Kit+ cells were transplanted per mouse. Mice analysed: BM Ctr, n=4, SLHR-iHP cells, n=20, SLHR-iHP kit+ cells, n=4. These are representative of three independent experiments. Scale bar, 2 mm. (b) Comparison of CFU-S12 potential of iHP cells induced by differing TF cocktails. SL-, SLB-, SLHR-, SLRH- or SLRB-iHP cells (tdTomato+) were transplanted into lethally irradiated SCID mice. Factors were delivered in pMXconstructs; SLHR denotes individual delivery of S, L, H and R; SLRH and SLRB denotes use of a polycistronic construct containing S, L and R in one pMX vector, together with individual delivery of either H or B in a separate construct. For SL-, SLB-, SLHR-iHP, 5 × 106 cells were transplanted. For SLRH- and SLRB-iHPs, 2 × 106 cells were transplanted. Mice analysed: for SL-, SLB-, SLHR-iHP, n=6, for SLRB- and SLRH-iHP, n=12 each. Scale bar: 2 mm. These are representative of three independent experiments. (c) Frequency of CFU-S12 of different iHP cells. iHP cells are named as in b. Data are shown as mean±s.d. per spleen. These data are from three independent experiments. (d) Representative FACS analysis of tdTomato+ cells in BM of SCID mice at 12–14 dpt. SLR factors were delivered in one polycistronic construct. For SLRB/H-iHP cells: 2 × 106 cells per mouse were transplanted; for Ctr BM cells (tdTomato+), 2 × 105 cells per mouse were transplanted. Percentage of tdTomato+ cells are shown as mean±s.d. (n=6 mice for each type of cells), representative from three independent experiments. (e) Representative FACS analysis of tdTomato+ cells stained with lineage markers (shown on the plots) in the BM of SCID mice transplanted with either SLRB/SLRH-HP cells and control BM cells (Ctr tdTomato+) at 12–14dpt. SLR factors were delivered in one polycistronic construct. Mice analysed: n=6 for each type of cells. Ery, Erythroid; Meg, Megakaryocytes. These data are representative of three independent experiments.

Mentions: Given that surface markers Kit and CD41 enrich for colony-forming progenitors in embryonic hematopoiesis34, we assessed the in vitro colony-forming activity of four cell populations (Kit+CD41−, Kit+CD41+, Kit−CD41+, Kit−CD41−) sorted from SLHR-iHP cultures (Supplementary Fig. 2f). Kit−CD41− cells only contained limited myeloid progenitor potential, while multipotent and committed progenitors were enriched in the Kit+ fraction (either CD41− or CD41+; Fig. 2g). Similarly in the collagen-based CFU-Mk assay, Kit+CD41+ progenitors could form CFU-mix containing both myeloid lineages (G/M) and megakaryocytes (Mk or Meg). By contrast, the Kit−CD41+ fraction harboured more committed megakaryocyte progenitor activity (Supplementary Fig. 2g). Collectively this demonstrates that oligopotent myeloerythroid progenitor activity is contained within Kit+ iHPs.


Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors
Runx1 together with HoxB4 or Bmi1 augments the CFU-S forming-ability of iHP cells.(a) Similar to control bone marrow cells (BM Ctr, tdTomato+), SLHR-iHP cells (tdTomato+) form tdTomato+ nodules in the spleen at 12 days post-transplantation (dpt) into lethally irradiated C57Bl/6 mice (CFU-S12). Factors were singly delivered in individual pMX constructs. 1 × 105 BM control cells, 5 × 106 SLHR-iHP cells or 1 × 106 Kit+ cells were transplanted per mouse. Mice analysed: BM Ctr, n=4, SLHR-iHP cells, n=20, SLHR-iHP kit+ cells, n=4. These are representative of three independent experiments. Scale bar, 2 mm. (b) Comparison of CFU-S12 potential of iHP cells induced by differing TF cocktails. SL-, SLB-, SLHR-, SLRH- or SLRB-iHP cells (tdTomato+) were transplanted into lethally irradiated SCID mice. Factors were delivered in pMXconstructs; SLHR denotes individual delivery of S, L, H and R; SLRH and SLRB denotes use of a polycistronic construct containing S, L and R in one pMX vector, together with individual delivery of either H or B in a separate construct. For SL-, SLB-, SLHR-iHP, 5 × 106 cells were transplanted. For SLRH- and SLRB-iHPs, 2 × 106 cells were transplanted. Mice analysed: for SL-, SLB-, SLHR-iHP, n=6, for SLRB- and SLRH-iHP, n=12 each. Scale bar: 2 mm. These are representative of three independent experiments. (c) Frequency of CFU-S12 of different iHP cells. iHP cells are named as in b. Data are shown as mean±s.d. per spleen. These data are from three independent experiments. (d) Representative FACS analysis of tdTomato+ cells in BM of SCID mice at 12–14 dpt. SLR factors were delivered in one polycistronic construct. For SLRB/H-iHP cells: 2 × 106 cells per mouse were transplanted; for Ctr BM cells (tdTomato+), 2 × 105 cells per mouse were transplanted. Percentage of tdTomato+ cells are shown as mean±s.d. (n=6 mice for each type of cells), representative from three independent experiments. (e) Representative FACS analysis of tdTomato+ cells stained with lineage markers (shown on the plots) in the BM of SCID mice transplanted with either SLRB/SLRH-HP cells and control BM cells (Ctr tdTomato+) at 12–14dpt. SLR factors were delivered in one polycistronic construct. Mice analysed: n=6 for each type of cells. Ery, Erythroid; Meg, Megakaryocytes. These data are representative of three independent experiments.
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f2: Runx1 together with HoxB4 or Bmi1 augments the CFU-S forming-ability of iHP cells.(a) Similar to control bone marrow cells (BM Ctr, tdTomato+), SLHR-iHP cells (tdTomato+) form tdTomato+ nodules in the spleen at 12 days post-transplantation (dpt) into lethally irradiated C57Bl/6 mice (CFU-S12). Factors were singly delivered in individual pMX constructs. 1 × 105 BM control cells, 5 × 106 SLHR-iHP cells or 1 × 106 Kit+ cells were transplanted per mouse. Mice analysed: BM Ctr, n=4, SLHR-iHP cells, n=20, SLHR-iHP kit+ cells, n=4. These are representative of three independent experiments. Scale bar, 2 mm. (b) Comparison of CFU-S12 potential of iHP cells induced by differing TF cocktails. SL-, SLB-, SLHR-, SLRH- or SLRB-iHP cells (tdTomato+) were transplanted into lethally irradiated SCID mice. Factors were delivered in pMXconstructs; SLHR denotes individual delivery of S, L, H and R; SLRH and SLRB denotes use of a polycistronic construct containing S, L and R in one pMX vector, together with individual delivery of either H or B in a separate construct. For SL-, SLB-, SLHR-iHP, 5 × 106 cells were transplanted. For SLRH- and SLRB-iHPs, 2 × 106 cells were transplanted. Mice analysed: for SL-, SLB-, SLHR-iHP, n=6, for SLRB- and SLRH-iHP, n=12 each. Scale bar: 2 mm. These are representative of three independent experiments. (c) Frequency of CFU-S12 of different iHP cells. iHP cells are named as in b. Data are shown as mean±s.d. per spleen. These data are from three independent experiments. (d) Representative FACS analysis of tdTomato+ cells in BM of SCID mice at 12–14 dpt. SLR factors were delivered in one polycistronic construct. For SLRB/H-iHP cells: 2 × 106 cells per mouse were transplanted; for Ctr BM cells (tdTomato+), 2 × 105 cells per mouse were transplanted. Percentage of tdTomato+ cells are shown as mean±s.d. (n=6 mice for each type of cells), representative from three independent experiments. (e) Representative FACS analysis of tdTomato+ cells stained with lineage markers (shown on the plots) in the BM of SCID mice transplanted with either SLRB/SLRH-HP cells and control BM cells (Ctr tdTomato+) at 12–14dpt. SLR factors were delivered in one polycistronic construct. Mice analysed: n=6 for each type of cells. Ery, Erythroid; Meg, Megakaryocytes. These data are representative of three independent experiments.
Mentions: Given that surface markers Kit and CD41 enrich for colony-forming progenitors in embryonic hematopoiesis34, we assessed the in vitro colony-forming activity of four cell populations (Kit+CD41−, Kit+CD41+, Kit−CD41+, Kit−CD41−) sorted from SLHR-iHP cultures (Supplementary Fig. 2f). Kit−CD41− cells only contained limited myeloid progenitor potential, while multipotent and committed progenitors were enriched in the Kit+ fraction (either CD41− or CD41+; Fig. 2g). Similarly in the collagen-based CFU-Mk assay, Kit+CD41+ progenitors could form CFU-mix containing both myeloid lineages (G/M) and megakaryocytes (Mk or Meg). By contrast, the Kit−CD41+ fraction harboured more committed megakaryocyte progenitor activity (Supplementary Fig. 2g). Collectively this demonstrates that oligopotent myeloerythroid progenitor activity is contained within Kit+ iHPs.

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