1. Volume 7, Issue 1, Pages 101-113 (July 2010)
An RNAi Screen Identifies Msi2 and Prox1 as Having Opposite Roles in the Regulation of Hematopoietic Stem Cell Activity 
Kristin J. Hope, Sonia Cellot, Stephen B. Ting, Tara MacRae, Nadine Mayotte, Norman N. Iscove, Guy Sauvageau 
Cell Stem Cell 
Volume 7, Issue 1, Pages (July 2010)
DOI: /j.stem
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Cell Stem Cell 2010 7, 101-113DOI: (10.1016/j.stem.2010.06.007)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
Identification of Polarity Proteins and Fate Determinants that Regulate Hematopoietic Repopulation
(A) Experimental design of primary screen and validation steps.
(B) Primary screen results at 20 weeks posttransplant. For constructs that achieved a gene transfer of 25%–75% (left), significant loss or accumulation of transduced cells was determined by normalizing the percent of GFP+ cells within the donor peripheral blood graft at 20 weeks to the corresponding percent GFP at day 0. Upper and lower thresholds (dashed lines) were established as the mean (black line) ± 1.5 standard deviations determined from multiple experiments with shLuciferase controls transduced over the 25%–75% range (see Figure S1B, Supplemental Experimental Procedures). In cases where gene transfer was >75% (right), values are shown as the percent GFP of the total peripheral blood. The mean from multiple shLuciferase experiments was 54% (black line) with a lower threshold value at 1.5 SD shown by the dashed line (see Figure S1B, Supplemental Data). Cases with gene transfer ratios between 65% and 75% were manually inspected for increases in output/input GFP ratios also occurring with at least one other independent shRNA.
(C) Q-RT-PCR assessment of relative transcript levels remaining in GFP+ cells sorted at day 0 to day 1 from two to three independent experiments. shRNAs achieving less than 40% transcript knockdown were eliminated from further study.
(D) Summary of the results of the primary and validation screens from candidate selection to final hit confirmation.
Data are presented as mean ± SEM. Asterisks indicate hits that fall above or below cutoffs. See also Figure S1 and Tables S1 and S2.
Cell Stem Cell 2010 7, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 2
Characterizing the Nature of Repopulation Impairment by Msi2, Pard6a, and Prkcz shRNAs
(A) Flow cytometric analysis of peripheral blood sampled from representative recipients of shLuciferase and shMsi2 transduced cells at 4 (middle) and 20 (right) weeks posttransplant. Gene transfer percentages assessed immediately after infection (D0) are also shown (left).
(B) Quantitative assessment of shRNA-induced defects in repopulation. shMsi2 and shPar6a results (49% and 51% gene transduction, respectively) are displayed as a ratio of the GFP% within the donor graft (at the indicated time point) to the GFP% at D0. Results for shRNAs to Prkcz are presented as %GFP of total peripheral blood because initial GFP% at D0 were consistently above 80% (6-shMsi2, n = 5; 3-shPar6a, n = 6; 1-shPrkcz, n = 4; 3 experiments each for 6-shMsi2 and 3-shPard6a, 2 experiments for 1-shPrkcz). In each case, the cumulative effect of each hairpin was tested against at least six mice transplanted with shLuciferase-transduced cells that were infected to similar levels on D0.
(C) Evaluation of repopulation after graded levels of Msi2 knockdown. Q-RT-PCR evaluation of Msi2 knockdown by four separate shRNAs (left). At 20 weeks posttransplant, repopulation (shown at right) decreased in direct proportion to knockdown levels (2-shMsi2, n = 6; 1-shMsi2, n = 5; 4-shMsi2, n = 5; 6-shMsi2, n = 5, except for 4-sh-Msi2 [2 experiments], results are shown from 3 separate experiments).
(D) Bright-field (left top) and fluorescence (left bottom) images of typical high proliferative potential (HPP) shLuciferase and shMsi2 GFP+ colonies (left). Colony-forming activity of GFP+-transduced cells plated immediately after infection (right) (n = 2 for each data set).
(E) Lineage distribution of colonies generated by shRNA-transduced progenitors (n = 2–4).
(F) Morphology of representative shLuciferase- and shMsi2-transduced cells after a 10-day culture period (left). Flow cytometric evaluation of the percentage of cells positive for the differentiation antigen Gr-1 (middle) (n = 3–10 for each shRNA). Day 10 CFC output of GFP+ cells (right) (n = 3–8 for each shRNA).
(G) Bone marrow homing efficiency of GFP+ shRNA-transduced progenitors at 24 hr posttransplant. Results are presented as the proportion of GFP+ colonies recovered from recipient bone marrow relative to the %GFP colonies input.
Total CFC output in (D) and (F) is shown as relative to corresponding untransduced cells. Values are represented as mean ± SEM. Asterisks indicate p values below See also Figures S2 and S3.
Cell Stem Cell 2010 7, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 3
Evaluation of the Role of Prox1 in Regulating HSC Numbers In Vivo
(A) Representative flow cytometric analysis of donor reconstitution in recipients of shLuciferase- and 2-shProx1-transduced cells at 4 (middle) and 20 (right) weeks posttransplant. D0 transduction efficiencies for each shRNA are also shown (left).
(B) Quantitative assessment of Prox1 shRNA-induced enhancement in repopulation (left). Results are shown in comparison to shLuciferase controls transduced over a similar range (data are summarized from 4 independent experiments: shLuciferase, n = 17; 2-shProx1, n = 7). Evaluation of knockdown in donor GFP+ and GFP− cells taken from two mice transplanted with 2-shProx1-transduced cells 35 weeks prior is shown at right.
(C) Percentages of donor GFP+ cells expressing B220, Gr-1, and CD3 in peripheral blood at 25–40 weeks posttransplant.
(D) Flow cytometry plots depicting the level of CD150+Sca+kit+ as a percentage of Lin−CD48− cells from GFP+ or GFP− bone marrow of representative recipients at 25 weeks posttransplant (left).
(E) Cumulative results illustrate the elevated percentage of Lin−CD150+CD48−Sca+kit+ cells within shProx1 GFP+ grafts as compared to GFP+ shLuciferase grafts or GFP− cells (shLuciferase GFP+, n = 3; GFP−, n = 12; 2-shProx1 GFP+, n = 2; 3-shProx1, n = 4).
Values are represented as mean ± SEM. ∗p < See also Figure S4.
Cell Stem Cell 2010 7, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 4
Determining the Potential for Prox1 Downregulation to Induce In Vitro HSC Expansion
(A) Morphological examination of transduced cells cultured for 7–10 days after infection with NupHD or shRNAs directed against Prox1 or Luciferase. Flow cytometric evaluation of the percentage of Gr-1−Sca+ cells within the transduced population of 7–10 day cultures (bottom right) (shLuciferase, n = 5; 3-shProx1, n = 5).
(B) Assessment of the in vivo repopulation contribution by 1/8th the contents of 7-day shProx1 or control shRNA 96-well cultures. Results are presented as the mean %GFP within the recipient peripheral blood at the indicated time points. Mean activity of stem cells (MAS) values calculated as outlined in Experimental Procedures (bone marrow cell culture, retroviral infection, and transplantation section) (shLuciferase, n = 8; 2-shProx1, n = 6; 3-shProx1, n = 3; HOXB4, n = 3) (two separate experiments for shLuc and 2-shProx1).
(C) Limiting dilution analysis of the HSC frequency in 7-day cultured 3-shProx1, shLuciferase, and HOXB4 cells. HSC frequencies are displayed beside the corresponding curve.
(D) Cell cycle analysis of GFP+ cells infected with shRNAs to Prox1 or Luciferase after 5 days of culture. Representative profiles are shown on the left while the quantitative assessment of the percentage of cells at different phases of the cell cycle is depicted on the right (shLuciferase, n = 3; 3-shProx1, n = 4; NupHD, n = 3).
Data are shown as mean ± SEM. Asterisks indicate p values below See also Table S3.
Cell Stem Cell 2010 7, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 5
Endogenous Expression of Msi2 and Effects on Gene Expression after Msi2 or Prox1 Modulation
(A) Q-RT-PCR analysis of Msi2 in the indicated stem and progenitor fractions isolated from two separate sorts. Msi2 levels were normalized to Gapdh.
(B) Q-RT-PCR evaluation of the expression level of Msi2 in control and NupPHD-overexpressing cells after 10 days of culture.
(C) Semiquantitative RT-PCR assessment of Msi2 transcript levels in the hematopoietic lineage hierarchy. Corresponding Gapdh levels are shown in Figure S5.
(D) Q-RT-PCR evaluation of expression changes of candidate target genes after Msi2 overexpression (left) and knockdown (right).
(E) Assessment of in vivo repopulation contribution by equivalent proportions of day-7 cultures initiated with HSCs overexpressing Msi2 or infected with control empty vector (MSCV). Results are presented as the mean %Ly5.1+ cells within the recipient peripheral blood at the indicated time points and are representative of data from seven mice per condition.
(F) Q-RT-PCR validation of the differential expression of targets, identified by microarray as being altered 2-fold in expression after Prox1 knockdown.
In (D) and (F), dashed green and red lines indicate 2-fold greater or decreased expression relative to control MSCV- (overexpression experiments) or shLuciferase- (knockdown experiments) transduced cells, respectively.
Data are shown as mean ± SEM. Asterisks indicate p values below See also Figure S5 and Table S4.
Cell Stem Cell 2010 7, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions