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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.


Generating induced hematopoietic progenitors from wild-type MEFs.(a) Schema of experimental design. MEFs (P0) were purified by sorting out any contaminating hematopoietic cells and passaged to P2-3 before experiments. iHP cells induced from MEFs by reprogramming factors were used for further characterization and evaluation; negative control cultures were transduced with empty vector (EV) only. (b) Representative ‘cobblestone' colonies at 24 dpi induced by lentiviral FuW-TetO vectors carrying 7F. Representative of three independent experiments. Scale bar: 100 μm. (c) CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, representative of three independent experiments. Scale bar: 100 μm for GM, 50 μm for GEMM and E colonies. (d) Frequency of different type of CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, with dox added (or withheld) at the beginning of CFC assays as indicated. Data are shown as mean±s.d. of four biological replicates from two independent experiments. (e) Representative ‘cobblestone' colonies (27 dpi) induced by different combinations of factors: SL, SLB, SLHR or SLBR (with factors singly delivered in individual pMX vectors), representative of three independent experiments. Scale bar: 100 μm. (f) Different types of representative CFU colonies derived from 27 dpi SLHR-iHP in CFC assays (factors were singly delivered in individual FuW-TetO vectors), representative of two independent experiments. Scale bar: 100 μm for GM/G colonies; 50 μm for GEMM/E/M/Mk colonies. (g) Frequencies of different types of colonies derived from FACS-sorted Kit+CD41−, Kit+CD41+, Kit−CD41+ and Kit−CD41− subsets of SLHR-iHP cells in CFC assays. Factors were singly delivered in individual FuW-TetO vectors. Data shown are mean±s.d. of biological triplicates. (h) Benzidine positive cells in a GEMM colony and adult (β-major) globin expression in CFU-E colonies as shown in f, representative of two independent experiments. BM: bone marrow cells. FL: E12.5 foetal liver cells. Data shown are mean±s.d. of technical triplicates. Scale bar, 100 μm. (i) Images of AchE+ megakaryocyte-containing colonies (CFU-mix and CFU-Mk) and phagocytic CD45+ cells, representative of three independent experiments. 27 dpi SLHR-iHPs (induced using pMX vectors) were used for CFU-MK assay. Scale bar, 200 μm for CFU-mix/Mk colonies; 100 μm for phagocytosis picture.
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f1: Generating induced hematopoietic progenitors from wild-type MEFs.(a) Schema of experimental design. MEFs (P0) were purified by sorting out any contaminating hematopoietic cells and passaged to P2-3 before experiments. iHP cells induced from MEFs by reprogramming factors were used for further characterization and evaluation; negative control cultures were transduced with empty vector (EV) only. (b) Representative ‘cobblestone' colonies at 24 dpi induced by lentiviral FuW-TetO vectors carrying 7F. Representative of three independent experiments. Scale bar: 100 μm. (c) CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, representative of three independent experiments. Scale bar: 100 μm for GM, 50 μm for GEMM and E colonies. (d) Frequency of different type of CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, with dox added (or withheld) at the beginning of CFC assays as indicated. Data are shown as mean±s.d. of four biological replicates from two independent experiments. (e) Representative ‘cobblestone' colonies (27 dpi) induced by different combinations of factors: SL, SLB, SLHR or SLBR (with factors singly delivered in individual pMX vectors), representative of three independent experiments. Scale bar: 100 μm. (f) Different types of representative CFU colonies derived from 27 dpi SLHR-iHP in CFC assays (factors were singly delivered in individual FuW-TetO vectors), representative of two independent experiments. Scale bar: 100 μm for GM/G colonies; 50 μm for GEMM/E/M/Mk colonies. (g) Frequencies of different types of colonies derived from FACS-sorted Kit+CD41−, Kit+CD41+, Kit−CD41+ and Kit−CD41− subsets of SLHR-iHP cells in CFC assays. Factors were singly delivered in individual FuW-TetO vectors. Data shown are mean±s.d. of biological triplicates. (h) Benzidine positive cells in a GEMM colony and adult (β-major) globin expression in CFU-E colonies as shown in f, representative of two independent experiments. BM: bone marrow cells. FL: E12.5 foetal liver cells. Data shown are mean±s.d. of technical triplicates. Scale bar, 100 μm. (i) Images of AchE+ megakaryocyte-containing colonies (CFU-mix and CFU-Mk) and phagocytic CD45+ cells, representative of three independent experiments. 27 dpi SLHR-iHPs (induced using pMX vectors) were used for CFU-MK assay. Scale bar, 200 μm for CFU-mix/Mk colonies; 100 μm for phagocytosis picture.

Mentions: In an effort to reprogram mouse embryonic fibroblasts (MEFs) into hematopoietic progenitors, we employed a reprogramming cocktail that included hematopoietic transcription factors with roles in either HSC specification or self-renewal. We started with a pool of seven well-characterized factors (henceforth, ‘7F'), namely Scl (S), Lmo2 (L), Runx1 (R), HoxB4 (H), Bmi1 (B), Gfi1 (G1) and Gata2 (G2)2627 in p53−/− MEFs, given indications that p53−/− MEFs are more easily reprogrammed into iPSCs28 or blood progenitors24. Fibroblasts that were depleted of hematopoietic cells (Fig. 1a) were co-infected with the combination of seven hematopoietic factors and then seeded on inactivated OP9 feeder cells29 (Fig. 1a). 14–16 days post infection (dpi), small clusters of round cells could be distinguished from the flat fibroblasts (Supplementary Fig. 1a). By day 24, clear ‘cobblestone areas' were observed (Supplementary Fig. 1a), bearing resemblance to cobblestone area-forming units previously described for hematopoietic stem/progenitor cells30. Within another week, rounded cells were released from the cobblestone areas in large numbers into the medium. These cobblestone colonies contained cells expressing hematopoietic stem/progenitor markers Kit and CD41 (refs 31, 32; Supplementary Fig. 1a). Suspension cells showed characteristics of hematopoietic cells by FACS and morphological analyses (Supplementary Fig. 1b).


Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors
Generating induced hematopoietic progenitors from wild-type MEFs.(a) Schema of experimental design. MEFs (P0) were purified by sorting out any contaminating hematopoietic cells and passaged to P2-3 before experiments. iHP cells induced from MEFs by reprogramming factors were used for further characterization and evaluation; negative control cultures were transduced with empty vector (EV) only. (b) Representative ‘cobblestone' colonies at 24 dpi induced by lentiviral FuW-TetO vectors carrying 7F. Representative of three independent experiments. Scale bar: 100 μm. (c) CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, representative of three independent experiments. Scale bar: 100 μm for GM, 50 μm for GEMM and E colonies. (d) Frequency of different type of CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, with dox added (or withheld) at the beginning of CFC assays as indicated. Data are shown as mean±s.d. of four biological replicates from two independent experiments. (e) Representative ‘cobblestone' colonies (27 dpi) induced by different combinations of factors: SL, SLB, SLHR or SLBR (with factors singly delivered in individual pMX vectors), representative of three independent experiments. Scale bar: 100 μm. (f) Different types of representative CFU colonies derived from 27 dpi SLHR-iHP in CFC assays (factors were singly delivered in individual FuW-TetO vectors), representative of two independent experiments. Scale bar: 100 μm for GM/G colonies; 50 μm for GEMM/E/M/Mk colonies. (g) Frequencies of different types of colonies derived from FACS-sorted Kit+CD41−, Kit+CD41+, Kit−CD41+ and Kit−CD41− subsets of SLHR-iHP cells in CFC assays. Factors were singly delivered in individual FuW-TetO vectors. Data shown are mean±s.d. of biological triplicates. (h) Benzidine positive cells in a GEMM colony and adult (β-major) globin expression in CFU-E colonies as shown in f, representative of two independent experiments. BM: bone marrow cells. FL: E12.5 foetal liver cells. Data shown are mean±s.d. of technical triplicates. Scale bar, 100 μm. (i) Images of AchE+ megakaryocyte-containing colonies (CFU-mix and CFU-Mk) and phagocytic CD45+ cells, representative of three independent experiments. 27 dpi SLHR-iHPs (induced using pMX vectors) were used for CFU-MK assay. Scale bar, 200 μm for CFU-mix/Mk colonies; 100 μm for phagocytosis picture.
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f1: Generating induced hematopoietic progenitors from wild-type MEFs.(a) Schema of experimental design. MEFs (P0) were purified by sorting out any contaminating hematopoietic cells and passaged to P2-3 before experiments. iHP cells induced from MEFs by reprogramming factors were used for further characterization and evaluation; negative control cultures were transduced with empty vector (EV) only. (b) Representative ‘cobblestone' colonies at 24 dpi induced by lentiviral FuW-TetO vectors carrying 7F. Representative of three independent experiments. Scale bar: 100 μm. (c) CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, representative of three independent experiments. Scale bar: 100 μm for GM, 50 μm for GEMM and E colonies. (d) Frequency of different type of CFU colonies derived from 27 dpi FuW-TetO-7F-induced iHP cells, with dox added (or withheld) at the beginning of CFC assays as indicated. Data are shown as mean±s.d. of four biological replicates from two independent experiments. (e) Representative ‘cobblestone' colonies (27 dpi) induced by different combinations of factors: SL, SLB, SLHR or SLBR (with factors singly delivered in individual pMX vectors), representative of three independent experiments. Scale bar: 100 μm. (f) Different types of representative CFU colonies derived from 27 dpi SLHR-iHP in CFC assays (factors were singly delivered in individual FuW-TetO vectors), representative of two independent experiments. Scale bar: 100 μm for GM/G colonies; 50 μm for GEMM/E/M/Mk colonies. (g) Frequencies of different types of colonies derived from FACS-sorted Kit+CD41−, Kit+CD41+, Kit−CD41+ and Kit−CD41− subsets of SLHR-iHP cells in CFC assays. Factors were singly delivered in individual FuW-TetO vectors. Data shown are mean±s.d. of biological triplicates. (h) Benzidine positive cells in a GEMM colony and adult (β-major) globin expression in CFU-E colonies as shown in f, representative of two independent experiments. BM: bone marrow cells. FL: E12.5 foetal liver cells. Data shown are mean±s.d. of technical triplicates. Scale bar, 100 μm. (i) Images of AchE+ megakaryocyte-containing colonies (CFU-mix and CFU-Mk) and phagocytic CD45+ cells, representative of three independent experiments. 27 dpi SLHR-iHPs (induced using pMX vectors) were used for CFU-MK assay. Scale bar, 200 μm for CFU-mix/Mk colonies; 100 μm for phagocytosis picture.
Mentions: In an effort to reprogram mouse embryonic fibroblasts (MEFs) into hematopoietic progenitors, we employed a reprogramming cocktail that included hematopoietic transcription factors with roles in either HSC specification or self-renewal. We started with a pool of seven well-characterized factors (henceforth, ‘7F'), namely Scl (S), Lmo2 (L), Runx1 (R), HoxB4 (H), Bmi1 (B), Gfi1 (G1) and Gata2 (G2)2627 in p53−/− MEFs, given indications that p53−/− MEFs are more easily reprogrammed into iPSCs28 or blood progenitors24. Fibroblasts that were depleted of hematopoietic cells (Fig. 1a) were co-infected with the combination of seven hematopoietic factors and then seeded on inactivated OP9 feeder cells29 (Fig. 1a). 14–16 days post infection (dpi), small clusters of round cells could be distinguished from the flat fibroblasts (Supplementary Fig. 1a). By day 24, clear ‘cobblestone areas' were observed (Supplementary Fig. 1a), bearing resemblance to cobblestone area-forming units previously described for hematopoietic stem/progenitor cells30. Within another week, rounded cells were released from the cobblestone areas in large numbers into the medium. These cobblestone colonies contained cells expressing hematopoietic stem/progenitor markers Kit and CD41 (refs 31, 32; Supplementary Fig. 1a). Suspension cells showed characteristics of hematopoietic cells by FACS and morphological analyses (Supplementary Fig. 1b).

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.