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The polycomb group protein L3MBTL1 represses a SMAD5-mediated hematopoietic transcriptional program in human pluripotent stem cells.

Perna F, Vu LP, Themeli M, Kriks S, Hoya-Arias R, Khanin R, Hricik T, Mansilla-Soto J, Papapetrou EP, Levine RL, Studer L, Sadelain M, Nimer SD - Stem Cell Reports (2015)

Bottom Line: Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells.We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation.Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pernaf@mskcc.org.

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KD of L3MBTL1 Primes the Hematopoietic Potential of iPSCs(A) Endogenous L3MBTL1 expression was assessed in iPSCs at different time points during the mesodermal, hematopoietic, and erythroid differentiation using quantitative real-time PCR. The data represent the mean ± SD of the three independent experiments.(B) Strategy diagram. L3MBTL1 expression is efficiently knocked down as assessed by western blot assay in undifferentiated iPSCs. Tubulin served as the loading control.(C) KD of L3MBTL1 increases the expression of mesodermal-specific transcription factors while it decreases expression of key markers of endodermal and ectodermal lineages, as shown in L3MBTL1-KD undifferentiated iPSCs by qPCR compared to controls. Data indicate the relative expression level of the gene of interest, normalized by GAPDH. The reported fold changes have been calculated by ΔΔCt analysis versus control cells. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(D) KD of L3MBTL1 increases CFU capacity of iPSC-derived HSCs. Cells from day 10 hEBs were plated in methylcellulose and colonies were scored after 15 days. The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(E) KD of L3MBTL1 promotes the emergence of early hemogenic precursor cells. EBs were harvested at day 10, prior to CD45 emergence, and analyzed by flow cytometry for CD31 and CD34 expression.Refer to Figure S1 for the characterization of the iPSC line generated from cord blood CD34+ cells.
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fig1: KD of L3MBTL1 Primes the Hematopoietic Potential of iPSCs(A) Endogenous L3MBTL1 expression was assessed in iPSCs at different time points during the mesodermal, hematopoietic, and erythroid differentiation using quantitative real-time PCR. The data represent the mean ± SD of the three independent experiments.(B) Strategy diagram. L3MBTL1 expression is efficiently knocked down as assessed by western blot assay in undifferentiated iPSCs. Tubulin served as the loading control.(C) KD of L3MBTL1 increases the expression of mesodermal-specific transcription factors while it decreases expression of key markers of endodermal and ectodermal lineages, as shown in L3MBTL1-KD undifferentiated iPSCs by qPCR compared to controls. Data indicate the relative expression level of the gene of interest, normalized by GAPDH. The reported fold changes have been calculated by ΔΔCt analysis versus control cells. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(D) KD of L3MBTL1 increases CFU capacity of iPSC-derived HSCs. Cells from day 10 hEBs were plated in methylcellulose and colonies were scored after 15 days. The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(E) KD of L3MBTL1 promotes the emergence of early hemogenic precursor cells. EBs were harvested at day 10, prior to CD45 emergence, and analyzed by flow cytometry for CD31 and CD34 expression.Refer to Figure S1 for the characterization of the iPSC line generated from cord blood CD34+ cells.

Mentions: The generation of iPSC lines has provided opportunities to understand the fundamental processes of human cell fate decisions in the context of tissue regeneration and human disease. We first observed spontaneous downregulation of L3MBTL1 expression in a human iPSC line generated from cord blood cells (iCBCs) (Figures S1A–S1C) upon mesodermal differentiation, suggesting that decreased L3MBTL1 expression is required for mesoderm specification (Figure 1A). To investigate the role of L3MBTL1 on the specification of hematopoiesis during iPSC differentiation, we knocked down L3MBTL1 using H1P-hygro-EGFP+ lentiviral vectors that express small hairpin RNAs (shRNAs) directed against L3MBTL1 (Figure 1B). The subcloned green fluorescent protein (GFP)+ colonies showed a marked decrease in L3MBTL1 expression and retained stem cell morphology and stem-cell-related cell surface markers, including TRA-1-81, KLF4, OCT4, and NANOG expression (Figures S1D–S1G). Expression of mesoderm-specific transcription factors (T, TAL1, LMO2, and RUNX1) was consistently increased in the KD cells, while key markers for endoderm and ectoderm decreased (Figure 1C). We observed downregulation of SOX2 even though the iPSCs maintained self-renewal properties. While consistent with a previous report (Wang et al., 2012), we did find upregulation of SOX3 (data not shown), which also could explain the self-renewal phenotype. Functional assessment of clonogenic hematopoietic progenitors (CFUs) revealed that the lack of L3MBTL1 induced an increase in hematopoietic progenitor cell generation (Figure 1D). The hematopoietic colonies derived from the L3MBTL1-KD cells were also larger in size (data not shown). CD45−CD31+CD34+ cells, which represent a primitive bipotent endothelial-like precursor population that is exclusively responsible for hematopoietic fate (Wang et al., 2004), were also much more abundant within the L3MBTL1-KD cells compared to controls (Figure 1E).


The polycomb group protein L3MBTL1 represses a SMAD5-mediated hematopoietic transcriptional program in human pluripotent stem cells.

Perna F, Vu LP, Themeli M, Kriks S, Hoya-Arias R, Khanin R, Hricik T, Mansilla-Soto J, Papapetrou EP, Levine RL, Studer L, Sadelain M, Nimer SD - Stem Cell Reports (2015)

KD of L3MBTL1 Primes the Hematopoietic Potential of iPSCs(A) Endogenous L3MBTL1 expression was assessed in iPSCs at different time points during the mesodermal, hematopoietic, and erythroid differentiation using quantitative real-time PCR. The data represent the mean ± SD of the three independent experiments.(B) Strategy diagram. L3MBTL1 expression is efficiently knocked down as assessed by western blot assay in undifferentiated iPSCs. Tubulin served as the loading control.(C) KD of L3MBTL1 increases the expression of mesodermal-specific transcription factors while it decreases expression of key markers of endodermal and ectodermal lineages, as shown in L3MBTL1-KD undifferentiated iPSCs by qPCR compared to controls. Data indicate the relative expression level of the gene of interest, normalized by GAPDH. The reported fold changes have been calculated by ΔΔCt analysis versus control cells. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(D) KD of L3MBTL1 increases CFU capacity of iPSC-derived HSCs. Cells from day 10 hEBs were plated in methylcellulose and colonies were scored after 15 days. The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(E) KD of L3MBTL1 promotes the emergence of early hemogenic precursor cells. EBs were harvested at day 10, prior to CD45 emergence, and analyzed by flow cytometry for CD31 and CD34 expression.Refer to Figure S1 for the characterization of the iPSC line generated from cord blood CD34+ cells.
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fig1: KD of L3MBTL1 Primes the Hematopoietic Potential of iPSCs(A) Endogenous L3MBTL1 expression was assessed in iPSCs at different time points during the mesodermal, hematopoietic, and erythroid differentiation using quantitative real-time PCR. The data represent the mean ± SD of the three independent experiments.(B) Strategy diagram. L3MBTL1 expression is efficiently knocked down as assessed by western blot assay in undifferentiated iPSCs. Tubulin served as the loading control.(C) KD of L3MBTL1 increases the expression of mesodermal-specific transcription factors while it decreases expression of key markers of endodermal and ectodermal lineages, as shown in L3MBTL1-KD undifferentiated iPSCs by qPCR compared to controls. Data indicate the relative expression level of the gene of interest, normalized by GAPDH. The reported fold changes have been calculated by ΔΔCt analysis versus control cells. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(D) KD of L3MBTL1 increases CFU capacity of iPSC-derived HSCs. Cells from day 10 hEBs were plated in methylcellulose and colonies were scored after 15 days. The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(E) KD of L3MBTL1 promotes the emergence of early hemogenic precursor cells. EBs were harvested at day 10, prior to CD45 emergence, and analyzed by flow cytometry for CD31 and CD34 expression.Refer to Figure S1 for the characterization of the iPSC line generated from cord blood CD34+ cells.
Mentions: The generation of iPSC lines has provided opportunities to understand the fundamental processes of human cell fate decisions in the context of tissue regeneration and human disease. We first observed spontaneous downregulation of L3MBTL1 expression in a human iPSC line generated from cord blood cells (iCBCs) (Figures S1A–S1C) upon mesodermal differentiation, suggesting that decreased L3MBTL1 expression is required for mesoderm specification (Figure 1A). To investigate the role of L3MBTL1 on the specification of hematopoiesis during iPSC differentiation, we knocked down L3MBTL1 using H1P-hygro-EGFP+ lentiviral vectors that express small hairpin RNAs (shRNAs) directed against L3MBTL1 (Figure 1B). The subcloned green fluorescent protein (GFP)+ colonies showed a marked decrease in L3MBTL1 expression and retained stem cell morphology and stem-cell-related cell surface markers, including TRA-1-81, KLF4, OCT4, and NANOG expression (Figures S1D–S1G). Expression of mesoderm-specific transcription factors (T, TAL1, LMO2, and RUNX1) was consistently increased in the KD cells, while key markers for endoderm and ectoderm decreased (Figure 1C). We observed downregulation of SOX2 even though the iPSCs maintained self-renewal properties. While consistent with a previous report (Wang et al., 2012), we did find upregulation of SOX3 (data not shown), which also could explain the self-renewal phenotype. Functional assessment of clonogenic hematopoietic progenitors (CFUs) revealed that the lack of L3MBTL1 induced an increase in hematopoietic progenitor cell generation (Figure 1D). The hematopoietic colonies derived from the L3MBTL1-KD cells were also larger in size (data not shown). CD45−CD31+CD34+ cells, which represent a primitive bipotent endothelial-like precursor population that is exclusively responsible for hematopoietic fate (Wang et al., 2004), were also much more abundant within the L3MBTL1-KD cells compared to controls (Figure 1E).

Bottom Line: Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells.We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation.Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pernaf@mskcc.org.

Show MeSH
Related in: MedlinePlus