Journal: Science Advances

Article Title: Regulation of eIF4E guides a unique translational program to control erythroid maturation

doi: 10.1126/sciadv.add3942

Figure Lengend Snippet: ( A ) HUDEP-2 cells transduced with retrovirus encoding eIF4E [pMSCV-eIF4E-IRES-GFP (green fluorescent protein)] or The RNAi Consortium (TRC) control (pMSCV-TRC-IRES-GFP) followed by GFP sorting, precursor expansion, and differentiation. ( B ) Representative flow cytometry plots comparing HUDEP-WT and HUDEP-eIF4E cells 4 days after induction of maturation. ( C to E ) Quantitation of flow cytometry–defined differences in maturation between WT and eIF4E HUDEP-2 cells by (C) CD71 and FSC, (D) CD34 and CD105, and (E) cKit. * P < 0.05, N = 3. ( F ) Representative Wright-Giemsa–stained cytospins of day 0 and +4 HUDEP-WT and HUDEP-eIF4E cells displaying relative homogeneity in day 0 of both conditions (top) and day +4 (bottom) showing marked anisocytosis in HUDEP-WT cells compared to HUDEP-eIF4E cells. Green arrowheads denote erythroblasts showing nuclear condensation and size restriction. Magnification, ×40. Black line indicates size marker of 100 μm. ( G ) Representative day +4 cell pellet of WT versus eIF4E HUDEP-2 cells showing impaired hemoglobinization evidenced by the pale color of the pellet. Statistical analysis between populations using paired t test compared to HUDEP-WT. N.S., not significant.

Article Snippet: Thawed cells were plated on retronectin-coated wells in CD34 + expansion media [StemSpan SFEM II, hSCF (100 ng/ml; Peprotech), Flt3 ligand (100 ng/ml; Peprotech), Thrombopoietin (TPO) (50 ng/ml; Peprotech), low-density lipoprotein (10 μg/ml; STEMCELL Technologies), UM171 (35 nM; Benchchem), and penicillin/streptomycin (Thermo Fisher Scientific)] for 48 hours of expansion before retroviral transduction with pMSCV-IRES-GFP or pMSCV-eIF4E-IRES-GFP as described above.

Techniques: Transduction, Flow Cytometry, Quantitation Assay, Staining, Marker

Journal: Science Advances

Article Title: Regulation of eIF4E guides a unique translational program to control erythroid maturation

doi: 10.1126/sciadv.add3942

Figure Lengend Snippet: ( A ) HUDEP-2 WT and eIF4E cells nucleofected with RNP containing Cas9 with crRNA:tracrRNA duplex specific to either noncoding negative control, PTPN6, or Igf2bp1. Single-cell clones were generated and expanded before induction of maturation. ( B ) Representative flow cytometry plots of CD71 versus FSC and CD34 versus CD105 expression demonstrating the reversal of erythroid maturation in HUDEP-eIF4E (negative CRISPR control) cells with simultaneous knockdown of PTPN6 (4E PTPN6 1 or 4E PTPN6 2) or Igf2bp1 (4E Igf2bp1 1 or Igf2bp1 2) in comparison to HUDEP-WT (negative CRISPR control). ( C ) Partial rescue of CD71 + erythroid maturation phases dependent on PTPN6 knockdown. * P < 0.05, N = 6. ( D ) Partial rescue of CD71 + erythroid maturation phases dependent on Igf2bp1 knockdown. * P < 0.05, N = 6. ( E ) Partial rescue of CD34 + , CD105 + and CD34 − , CD105 + populations with knockdown of PTPN6 or Igf2bp1. * P < 0.05, N = 6. Statistical analysis between populations calculated using paired t test compared to HUDEP-4E negative control (4E Ng Cnt).

Article Snippet: Thawed cells were plated on retronectin-coated wells in CD34 + expansion media [StemSpan SFEM II, hSCF (100 ng/ml; Peprotech), Flt3 ligand (100 ng/ml; Peprotech), Thrombopoietin (TPO) (50 ng/ml; Peprotech), low-density lipoprotein (10 μg/ml; STEMCELL Technologies), UM171 (35 nM; Benchchem), and penicillin/streptomycin (Thermo Fisher Scientific)] for 48 hours of expansion before retroviral transduction with pMSCV-IRES-GFP or pMSCV-eIF4E-IRES-GFP as described above.

Techniques: Negative Control, Clone Assay, Generated, Flow Cytometry, Expressing, CRISPR

Journal: Nature Communications

Article Title: Adult stem cell deficits drive Slc29a3 disorders in mice

doi: 10.1038/s41467-019-10925-3

Figure Lengend Snippet: ENT3 is essential for self-renewal. MSC clonogenicity when subjected to serial passage ( n = 6, mean ± SEM) ( a ). Percent cells expressing MSC markers after each passage of serial cloning ( n = 6, mean ± SEM) ( b ). Culture expansion capacity of HSCs ( n = 3, mean ± SEM) ( c ). Percent of cells expressing HSC markers after each passage ( n = 6, mean ± SEM) ( d ). Expression of Wnt and Notch targets and pluripotency (pluri) marker genes in Slc29a3 −/− MSCs and HSCs ( n = 6, mean ± SEM) ( e ). HSCs and MSCs were derived from 12-weeks-old animals ( a – e ). Cellularity ( n = 6, mean ± SEM), MSC CFU-F ( n = 6, mean ± SEM) and LSK FLT3 − CD34 + ( n = 7, mean ± SEM) frequencies in bone marrow measured in different age groups (). MNC, mononuclear cells; CFU-F, CFU-Fibroblast ( f ). Ability of HSCs (1 × 10 4 cells) to rescue radiation lethality in Slc29a3 −/− mice after transplantation presented as Kaplan–Meier survival curves ( n = 6/group, *** P < 0.001; Mantel-Cox test) ( g ) and post transplantation bone marrow cellularity ( n = 6, mean ± SEM) ( h ). Ability of Slc29a3 −/− HSCs (1 × 10 4 cells), Slc29a3 −/− MSCs (5 × 10 5 cells), alone or combined, to rescue radiation lethality in Slc29a3 −/− mice after the expression of RFP or ENT3 presented as Kaplan–Meier survival curves, n = 6/group, * P < 0.05, ** P < 0.01, *** P < 0.001; Mantel-Cox test ( i ) and post transplantion bone marrow cellularity ( n = 6/group, * P < 0.05; Mantel-Cox test) ( j ). Relative expression of ENT3 in MSCs and HSCs transduced with a lentiviral construct harboring RFP or ENT3 compared with expression in WT mice ( k ). HSCs and MSCs were derived from 8-weeks-old animals ( g – k ). Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons post-test and two-tailed Student’s t- test. * P < 0.05. Source data are provided as a Source Data file. blue circles, Slc29a3 +/+ ; magenta squares, Slc29a3 −/−

Article Snippet: HSCs were maintained in HSC medium [serum-free stem cell expansion medium (StemSpan, Stem Cell Technologies) supplemented with Stem Cell Factor (SCF) (100 ng/ml)(R&D systems)] for downstream applications.

Techniques: Expressing, Cloning, Marker, Derivative Assay, Transplantation Assay, Transduction, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: Adult stem cell deficits drive Slc29a3 disorders in mice

doi: 10.1038/s41467-019-10925-3

Figure Lengend Snippet: ENT3 loss impairs induction of autophagic response. Representative immunoblots of LC3 and p62 protein expression in MSCs and HSCs under glucose starvation (1 h; above) followed by treatment with vehicle ((−) BAF) or bafilomycin A1 (( + )BAF; 100 nM, 4 h) under glucose starvation (below). β-Actin served as the loading control ( a ). Representative fluorescent images (above) and quantification (below) of LC3 puncta formation (green) in MSCs and HSCs under glucose starvation (1 h) followed by treatment with vehicle ((−) BAF) or bafilomycin A1 (( + )BAF; 100 nM, 4 h) under glucose starvation. Original magnification, × 60; Scale bar: 10 μm. Nuclei stained with DAPI. ( n = 3, mean ± SEM) ( b ). TEM analysis of autophagosome (yellow arrowhead) and lysosome (yellow arrow), mitochondria (red arrowheads), and ER (red arrow) in Slc29a3 +/+ and Slc29a3 −/− MSCs. Insets are expanded (right). Scale bar: 2 μm. N, nucleus ( c ). Effect of RAPA (rapamycin) treatment (0.5–2 µM) on GFM-induced MSC and HSC survival ( n = 4, mean ± SEM) and death ( n = 6, mean ± SEM) as assayed by MTT assay and active caspase 3 measurement, respectively. The ‘0’’ concentration compares Slc29a3 −/− cell survival with Slc29a3 +/+ cells and ‘0.5–2’’ µM compares Slc29a3 −/− cell survival with Slc29a3 +/+ cells in the presence of respective RAPA concentrations. HSCs and MSCs were derived from 12-weeks-old mice ( d ). Effect of ATG7 (ATG7-OE) or ENT3 (ENT3-OE) overexpression on glucose starvation-induced MSC survival ( n = 8, mean ± SEM) ( e ). Relative expression of ENT3 and ATG7 in Slc29a3 −/− MSCs and HSCs transduced with lentiviruses harboring RFP or ENT3 compared with expression in Slc29a3 +/+ (WT) mice ( n = 3, mean ± SEM) ( f ). Effect of RAPA (0.5 µM), ATG7-OE or ENT3-OE on osteogenic medium (3 days)-induced transcription factor and marker gene expression in Slc29a3 −/− MSCs ( n = 6, mean ± SEM) ( g ). Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons post-test and two-tailed Student’s t- test. * P < 0.05. All phenotypes were assessed in MSCs and HSCs derived from 12-week-old mice. GFM, glucose-free medium. Source data are provided as a Source Data file

Article Snippet: HSCs were maintained in HSC medium [serum-free stem cell expansion medium (StemSpan, Stem Cell Technologies) supplemented with Stem Cell Factor (SCF) (100 ng/ml)(R&D systems)] for downstream applications.

Techniques: Western Blot, Expressing, Control, Staining, MTT Assay, Concentration Assay, Derivative Assay, Over Expression, Transduction, Marker, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Adult stem cell deficits drive Slc29a3 disorders in mice

doi: 10.1038/s41467-019-10925-3

Figure Lengend Snippet: ENT3 activates the AMPK signaling pathway. Representative immunoblots of proteins involved in the AMPK-mTOR-ULK axis in MSCs and HSCs derived from 12-week-old mice under basal, glucose-starved (GFM), and AICAR- or rapamycin (RAPA)-treated conditions ( a ). Immunoblot analysis of the AMPK-mTOR-ULK axis in HEK293-expressing control-shRNA, ENT3-shRNA, pE-YFP, or pEYFP-ENT3 subjected to GFM ( b ). Immunoblotting and quantification of the ENT3 shRNA inhibition of AMPK phosphorylation and reversal by ENT3YFP in HEK293 ( c ). Effect of AICAR on GFM-induced MSC and HSC survival and death as assayed by MTT assay and active caspase 3 measurement, respectively ( n = 6, mean ± SEM). The ‘0’’ concentration compares Slc29a3 −/− cell survival with Slc29a3 +/+ cells and ‘100–500’’ µM compares Slc29a3 −/− cell survival with Slc29a3 +/+ cells in the presence of respective AICAR concentrations ( n = 4, mean ± SEM). HSCs and MSCs were derived from 12-week-old mice ( d ). Effect of AICAR (500 µM) on osteogenic medium (3 days)-induced transcription factors (TF) and marker genes expression ( n = 6, mean ± SEM) in Slc29a3 −/− MSCs. HSCs and MSCs were derived from 12-week-old mice ( e ). Immunostaining analysis of AMPK (green) and mTOR (green) in Slc29a3 −/− and Slc29a3 +/+ MSCs (derived from 12-week-old mice). Original magnification, × 60; Scale bar: 10 μm ( f ). Immunoblot analysis of pmTOR, pAMPK, LAMP1 (lysosomal marker), lamin B1 (nuclear marker), and tubulin 1 (cytoplasmic marker) in lysates prepared from purified lysosomal fraction (PLF) isolated from Slc29a3 −/− and Slc29a3 +/+ MSCs (derived from 12-week-old mice) ( g ). WT and AMPK KO (α1/α2 −/− ) MEFs were transfected with mCherry-LC3 and pEYFP-hENT3 plasmids and LC3 and YFP fluorescence were visualized and quantified ( n = 3, mean ± SEM). Scale bar: 10 μm. Immunoblots of AMPK, pAMPK, and LC3 forms in WT and AMPK KO MEFs transduced with AAV harboring GFP (-) or mENT3 ( + ) (right) ( h ). GFM, glucose-free medium. Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons post-test and two-tailed Student’s t- test. * P < 0.05. Source data are provided as a Source Data file

Article Snippet: HSCs were maintained in HSC medium [serum-free stem cell expansion medium (StemSpan, Stem Cell Technologies) supplemented with Stem Cell Factor (SCF) (100 ng/ml)(R&D systems)] for downstream applications.

Techniques: Western Blot, Derivative Assay, Expressing, Control, shRNA, Inhibition, Phospho-proteomics, MTT Assay, Concentration Assay, Marker, Immunostaining, Purification, Isolation, Transfection, Fluorescence, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: Adult stem cell deficits drive Slc29a3 disorders in mice

doi: 10.1038/s41467-019-10925-3

Figure Lengend Snippet: AICAR and SCT improve survival and alleviate dysfunction in Slc29a3 −/− mice. AICAR injection (500 mg/kg; SID) ( n = 10/group) and SCT (1 × 10 4 Slc29a3 +/+ HSCs and 5 × 10 5 MSCs) ( n = 16/group) extend the survival of Slc29a3 −/− mice (*** P < 0.001; Mantel-Cox test) ( a ). AICAR-treated surviving mice show facial alopecia (insets; below), while both the AICAR and SCT groups show improved appearance and medullary hematopoiesis (right) ( b ). Changes in body weight ( c ), EchoMRI-measured fat and lean mass ( d ), absolute parametrial (PM) and inguinal (ING) fat pad mass ( e ), absolute soleus (SOL) and gastrocnemius (GA) skeletal muscle (SKM) mass ( f ) and bone marrow CFU-F frequency ( g ) in AICAR-treated surviving mice and SCT mice ( n = 6, mean ± SEM). Clonogenicity ( h ) and marker expression ( i ) with serial passage of MSCs derived from SCT mice ( n = 6, mean ± SEM). mRNA expression of transcription factors and markers after 14 (osteoblasts and adipocytes) and 28 (myocytes and chondrocytes) days of the differentiation of MSCs derived from SCT mice ( n = 6, mean ± SEM) ( j ). Hematological parameters in AICAR-treated surviving mice and SCT mice. AICAR-treated surviving Slc29a3 −/− mice ( n = 4, mean ± SEM) and SCT mice ( n = 7, mean ± SEM) at 28 weeks are compared with saline-treated Slc29a3 −/− ( n = 5, mean ± SEM) and WT mice ( n = 6, mean ± SEM) at 12 weeks ( k ). Frequency of erythroid subpopulations within the bone marrow of AICAR-treated surviving mice and SCT mice ( n = 6, mean ± SEM) ( l ). Cellularity (above) and HSC frequencies (below) in AICAR-treated ( n = 5, mean ± SEM) and SCT mouse bone marrow ( n = 7, mean ± SEM) ( m ). Culture expansion capacity ( n ) and percent of cells expressing HSC markers ( o ) with serial passage of SCT mouse bone marrow-derived HSCs ( n = 6, mean ± SEM). CFU-forming capacity (above) and mRNA expression of transcription factors and markers (below) after 14 days of HSC differentiation in SCT mice ( n = 6, mean ± SEM) ( p ). SCT, stem cell transplantation; CFU, colony-forming unit; CFU-F, CFU-Fibroblast; Hb, hemoglobin; PLT, platelet; LY, lymphocyte; MO, monocyte; NE, neutrophil; EO, eosinophil; ProE, proerythroblasts; EryA, early basophilic erythroblasts; EryB, late basophilic and polychromatic erythroblasts; EryC, orthochromatic erythroblasts/reticulocytes; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; BFU-E, burst-forming unit-erythroid, colony-forming unit, CFU; CFU-M, CFU-macrophage, CFU-G, CFU-granulocyte; CFU-GM, CFU-granulocyte, macrophage. Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons post-test and two-tailed Student’s t -test. * P < 0.05. Source data are provided as a Source Data file

Article Snippet: HSCs were maintained in HSC medium [serum-free stem cell expansion medium (StemSpan, Stem Cell Technologies) supplemented with Stem Cell Factor (SCF) (100 ng/ml)(R&D systems)] for downstream applications.

Techniques: Injection, Marker, Expressing, Derivative Assay, Saline, Transplantation Assay, Two Tailed Test

Journal: Blood Advances

Article Title: M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion

doi: 10.1182/bloodadvances.2018015685

Figure Lengend Snippet: M1-MΦs and M2-MΦs have opposite effects on HSC self-renewal and expansion in vitro. (A) Diagram illustrating the design of the experiments in panel B, in which mouse BM LSK cells were cultured with sorted mouse BM CD115+Gr-1high Mos (Gr-1high Mo), CD115+Gr-1low Mos (Gr-1low Mo), or CD115−Gr-1lowF4/80+SSClow MΦs (MΦ). (B) Numbers of total cells, LSK cells, and 5-week CAFCs in the input LSK cells and the progeny from various cultures shown in panel A. aP < .05 vs Input, bP < .05 vs Input and without MΦs (W/O MΦ), cP < .05 vs all other groups, dP < .05 vs all other groups except Input. (C) Diagram illustrating the design of the experiments in panel D. MΦs were isolated from mouse BM (BM-MΦs) or peritoneal cavity (P-MΦ). (D) Numbers of total cells, LSK cells, and 5-week CAFCs in the input LSK cells and the progeny from various cultures shown in panel C. aP < .05 vs W/O MΦ, bP < .05 vs Input and W/O MΦ, cP < .05 vs all other groups. (E) Diagram illustrating the experimental design for peritoneal MΦ polarization and LSK cell cocultures. (F) Relative gene expression in M1-MΦs and M2-MΦs compared with MΦs analyzed by quantitative polymerase chain reaction. *P < .05, **P < .01, and ***P < .001 vs cells cultured with M1-MFs; unpaired Student t test. (G) Arg1 activity in MΦs, M1-MΦs, and M2-MΦs. aP < .05 vs MΦ, bP < .05 vs MΦ and M1-MΦ. (H) Numbers of total cells, LSK cells, and 5-week CAFCs in the input LSK cells and the progeny from various cultures shown in panel E. aP < .05 vs Input, bP < .05 vs MΦ, cP < .05 vs Input and W/O MΦ, dP < .05 vs Input, W/O MΦ, and MΦ, eP < .05 vs all other groups, fP < .05 vs all other groups except MΦs. All data are mean ± standard error of the mean (SEM) (n = 3 independent cultures) and were analyzed by 1-way analysis of variance (ANOVA).

Article Snippet: Lin − Sca1 + c-kit + (LSK) cells (2 × 10 3 /mL per well in a 12-well plate) were cultured in a mouse HSC expansion medium (StemSpan medium; STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 20 ng/mL recombinant mouse thrombopoietin (TPO), and stem cell factor (SCF) (PeproTech), with or without Mos or various MΦs (1×10 5 per well), as shown in .

Techniques: In Vitro, Cell Culture, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Blood Advances

Article Title: M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion

doi: 10.1182/bloodadvances.2018015685

Figure Lengend Snippet: M1-MΦs and M2-MΦs differentially regulate HSC self-renewal and in vitro expansion via NOS2 and Arg-1, respectively. (A) Nos2 knockout abrogates the inhibitory effect of M1-MΦs on 5-week CAFCs. LSK cells (2 × 103) were cocultured with 1 × 105 M1-MΦs from wild-type mice and Nos2 knockout (Nos2−/−) mice in StemSpan medium supplemented with 20 ng/mL SCF and TPO for 6 days. Numbers of total cells, LSK cells, and 5-week CAFCs in the input LSK cells and the progeny from these cultures are presented as mean ± SEM (n = 3 independent cultures). aP < .05 vs Input, bP < .05 vs M1-MΦ, unpaired Student t test. (B) Arg1 knockout abrogates the promoting effect of M2-MΦs on 5-week CAFCs. LSK cells were cocultured with M2-MΦs from wild-type mice and Arg1-knockout (Arg1−/−) mice, as described in panel A. The data are mean ± SEM (n = 3 independent cultures). aP < .05 vs Input, bP < .05 vs Input and M2-MΦ, unpaired Student t test. (C) Spermidine dose dependently increases the expansion of LSK cells and 5-week CAFCs in vitro. LSK cells were cultured with increasing concentrations of spermidine, as described in panel A. Data are mean ± SEM (n = 3 independent cultures). aP < .05 vs Input, bP < .05 vs 0 µM, cP < .05 vs 1 µM, dP < .05 vs 5 µM, 1-way ANOVA. (D) Relative gene expression in LSK cells sorted from the progeny of LSK cells cultured with M1-MΦs and M2-MΦs compared with that of input LSK cells revealed that coculture with M2-MΦs upregulated the expression of several HSC self-renewal and antiapoptotic genes, whereas coculture with M1-MΦs had opposite effects and increased the expression of the proapoptotic protein Bax. Data are mean ± SEM (n = 3 independent cultures). *P < .05, ***P < .001 vs cells cultured with M1-MΦs, unpaired Student t test. (E) Representative flow cytometric analysis of apoptosis (left panel) and percentage of apoptotic cells (right panel) in input LSK cells and LSK cells after culture with M1-MΦs or M2-MΦs or without MΦs. Data are mean ± SEM (n = 2 independent cultures). ***P < .001 between the cells cultured with M1-MΦs and all other cells, unpaired Student t test. (F) Hypothetical model illustrating the role of MΦ polarization in the regulation of mouse BM HSC self-renewal and expansion in vitro.

Article Snippet: Lin − Sca1 + c-kit + (LSK) cells (2 × 10 3 /mL per well in a 12-well plate) were cultured in a mouse HSC expansion medium (StemSpan medium; STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 20 ng/mL recombinant mouse thrombopoietin (TPO), and stem cell factor (SCF) (PeproTech), with or without Mos or various MΦs (1×10 5 per well), as shown in .

Techniques: In Vitro, Knock-Out, Cell Culture, Gene Expression, Expressing