Attenuation of miR-126 Activity Expands HSC In Vivo without Exhaustion

Summary Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.

. Generation of tools for stable miR-126 gain-and loss-of-function in human cells.
This data is directly relevant to Figure 1 because it provides biological validation of the lentivirus tools that are used for enforced expression and knockdown of miR-126 in human and murine HSC. This data provides further support for the clonogenic data and lineage differentiation presented in Figure  2.

Figure S3. miR-126 KD in murine HSPC in vivo
This data provides further support to Fig. 3, where knockdown of miR-126 increases long-term or serial engraftment. Furthermore, this data shows that the effects are similar in strain swap transplantation experiments and provide evidence that the in vivo effects of miR-126 KD were not due to the outgrowth of a single aberrant clone.  This data provides SILAC proteomic analysis of K562 cells after 126/KD generating another unbiased approach to predict the pathways perturbed by miR-126 in Figure 5. In addition, we present further western blot validation of the PI3K/AKT/GSK3 pathway. This data supports our hypothesis that SCF signals through PI3K/AKT in Figure 6.        Figure S6 -Related to Figure 6 miRNA Control of HSC Quiescence Figure S1. Generation of tools for stable miR-126 gain-and loss-of-function in human cells.

SUPPLEMENTAL FIGURE LEGENDS
(A) miR-126 activity in K562 cells as compared to the indicated CD34 + huCB subpopulations.
Cells were transduced with a miR-126 reporter BdLV or a control BdLV, and miRNA activity was calculated as previously described (Gentner et al., 2010). Results are shown as mean ± sem of n=3 independent experiments.
(B) Expression of miR-126 in K562 cells transduced with increasing doses of the 126KD (red) and 126OE LV (blue) relative to CTRL LV transduced cells. miR-16 was used as a normalizer.
The average vector copy number (VCN) per genome is indicated on the x axis. Other miRNAs investigated, such as let7a and miR-142, were unchanged upon 126KD or 126OE (data not shown). Results are shown as mean ± sem of n=3 technical replicates.
(C) Representative western blots for PI3K p85 and SPRED1, two previously validated direct miR-126 targets (Fish et al., 2008;Guo et al., 2008;Wang et al., 2008) (G) Effect of 126OE on growth of CD34 + CD38 -huCB cells in erythrocyte promoting culture conditions. Results are shown as mean ± sem of n=3 independent experiments. A 3.8-fold reduction in total cell output upon 126OE was observed. However, constitutive expression of miR-126 had no effect on megakaryocytic differentiation (data not shown), suggesting that high levels of miR-126 in erythroid progenitors may stimulate differentiation toward megakaryocytes.
This data matches our colony data from early cytokine culture where 126OE decreased overall colony number and 126KD increased overall number, with the most significant differences being in the erythroid and mixed colonies (see Figure 2c-e).
(H) Effect of 126OE on development of erythroid human graft in the xenograft environment.     Results represent the mean ± sem of n=7 mice per group. Please note that -in agreement with previous experiments shown in Figure 4G, 126OE chimerism was significantly reduced in HSC and in LSK progenitors, while chimerism of CTRL vector transduced cells was not significantly different between these populations. * p < 0.05, ** p < 0.01. in the SILAC experiment as highlighted (orange indicates up regulation, while green indicates down regulation). PI3Kp85b and AKT2 down regulation upon 126OE were confirmed by western blot (see Figure S1D and Figure S5D) while SILAC quantification did not reach   CD34 + huCB cells were starved for 3h in cytokine free medium and then stimulated with the indicated cytokine alone or the TSF6 combination (see Experimental procedures).
Histograms show the MFI of the stimulated sample (grey filled area), non-stimulated control (black line) and a cytokine stimulated, Wortmannin treated sample (silver line). Percentages refer to the stimulated cells falling into the positive gate, the number below denotes the MFI of Phospho-AKT (Ser473) positive cells.

Development of Lentiviral Tools for Stable miR-126 Gain-and Loss-of-Function Studies
In order to study the function of miR-126 in HSPC, we developed lentiviral vectors for stable overexpression (gain-of-function approach) or antagonism of miR-126 (loss-of-function or knock-down approach). Both vectors exploit the strong spleen focus forming virus (SFFV) promoter and couple miR-126 up-or down-modulation with the expression of a transcriptionally linked (co-transcribed) fluorescence reporter. In the over-expressing vector (126OE vector) , the pri-mir-126 sequence is cloned within an intron upstream of the reporter. In the "knockdown" or "sponge" vector (126KD) (Gentner et al., 2008) the reporter transcript contains 8 tandem copies of an imperfectly complementary miR-126 target sequence within the 3' untranslated region (UTR) (Fig. 1c). If not otherwise mentioned, vectors containing the empty intron sequence or scrambled target sequences not complementary to any known miRNA served as controls for 126OE or 126KD, respectively.
Vectors were first tested in K562 cells that naturally express miR-126 to a similar extent as CD34 + CD38 -HSPC (Fig. S1a). Transduction with the 126OE vector led to a clear dosedependent increase (up to 8-fold) in miR-126 levels, whereas the 126KD vector led to 2-fold decrease in miR-126 levels (Fig. S1b), likely due to increased target-mediated turnover. This is not, however, the main mechanism of action of the KD vector, which inhibits miRNA activity by expressing excess miRNA targets that compete with the endogenous ones. Neither vector detectably perturbed the expression of unrelated miRNAs ( Fig. S4A and data not shown). We then determined the expression of previously reported miR-126 targets including sprouty-related EVH1 domain containing 1 (SPRED1) and phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) (Fish et al., 2008;Guo et al., 2008;Wang et al., 2008) by Western blot and qPCR ( Fig. S1c-d). PIK3R2 but not SPRED1 was responsive to 126OE or 126KD in K562 cells, revealing a vector dose-dependent, 1.5-to 2-fold down-or up-regulation, respectively, as compared to CTRL vector-transduced cells, both at the protein and mRNA level. PIK3R2 upregulation by increasing doses of 126KD was paralleled by a disproportionate increase in the expression of the fluorescence reporter as compared to the lower doses. This surge in fluorescence is expected because the reporter transcript carries miR-126 target sequences in the 3' UTR and its expression is subjected to miR-126 suppression until the target or "sponge" sequence reaches sufficient concentration to saturate miR-126 activity (Fig. S1e). Thus, the reporter expression level provides a surrogate marker to identify the cells in which miR-126 has been knocked down most effectively (Gentner et al., 2008). Knock-down of miR-126 was clearly detectable in the bulk population at ≥ 8 average vector copies per cell, a realistic goal that can be achieved in primary hematopoietic cells using an optimized transduction protocol (Santoni de Sio and Naldini, 2009;Biffi et al., 2011). These data show that stable 126OE and 126KD are feasible in a cell line resembling human HSPC in terms of miR-126 expression level, without evidence of deregulation of miRNA processing or other types of toxicity.
Using an optimized transduction protocol, we consistently achieved >90% transduction of CD34 + lin-CB cells in cytokine-supplemented serum-free medium and up to 10 average vector integrations per cell (Fig. S1f). The degree of miR-126 up-or down-modulation was functionally relevant, as the expression levels of the previously validated target PIK3R2 were specifically and significantly decreased upon overexpression, and increased upon knockdown (Fig. 1d).

miR-126 modulation alters megakaryocyte/erythrocyte differentiation
In suspension cultures designed to promote erythrocyte and megakaryocyte differentiation, we noted increased erythroid and megakaryocytic differentiation of CD34 + CD38lin-CB cells upon 126KD and a strong inhibition of erythropoiesis, upon 126OE ( Fig. S2c-h). Interestingly, the effects on erythroid and megakaryocyte differentiation must occur in a primitive cell type, since 126KD in flow purified MEP populations displayed no alterations in differentiation potential.

In vitro effects of miR-126 modulation are conserved in murine cells
To assess whether the regulatory function of miR-126 was conserved across species, we performed liquid culture and colony forming assays upon 126KD or 126OE in sorted lineage depleted (Lin -) Kit + murine HSPC. Cell cycle analysis gated on highly transduced Sca1 + cells showed an increase in S/G 2 /M and a decrease in G 0 upon 126KD, with an opposing behavior upon 126OE (Fig. S2i). Colony forming potential was significantly increased after re-plating of 126KD cells, and reduced upon 126OE (Fig. S2j). Of note, Annexin V staining showed negligible levels of apoptosis in all treatment groups (Fig. S2k).

126OE Phenotype is not Dependent on Artificially High Expression Levels
To examine the effect of miR-126 over-expression on the fate of murine HSCs in vivo, we performed competitive murine transplantation experiments. Lin -BM cells from congenic mice (Ptprc a and Ptprc b , respectively) were transduced with a 126OE vector or with a CTRL vector, and injected in a 1:1 ratio into myeloablated recipients. qPCR analysis performed on peripheral blood cells from mice engrafted with HSPC transduced with the 126OE vector based on the SFFV promoter (see main text and Fig.4e-h) demonstrated that OFP marker positive cells ectopically expressed miR-126 to similar levels as miR-16, one of the most abundant endogenous miRNAs (Fig. S4a). To exclude the possibility that the observed phenotype was dependent on toxicity due to very high levels of miR expression, we repeated the competitive transplantation experiments with ectopically expressing miR-126 from the weaker EF1 promoter. This vector resulted in a moderate level of overexpression of miR-126 in the peripheral blood of mice, to levels similar to the ubiquitously expressed miRNA let-7a (Fig.   S4b). Importantly, all aspects of the 126OE phenotype (initial burst followed by progressive reduction in 126OE cells) were faithfully reproduced (Fig. S4c).

Impact of 126OE on the early engraftment phase
Serial complete blood counts (CBC) performed every 3-4 days during the engraftment phase revealed a significant advantage of mice transplanted with 126OE cells in platelet and leukocyte recovery which was, however, limited to a short time window (Fig. S4d). The duration of severe neutropenia (ANC<500/l) and thrombocytopenia (<5x10 4 /l) was significantly shortened in mice transplanted with 126OE cells (Fig. S4d). The differences in engraftment kinetics were not due to a difference in homing capacity of 126OE cells, since mice had, in comparison to the control groups, equal or slightly reduced grafts in the BM at 3 days post infusion (Fig. S4e).
These experiments suggest that, initially after transplantation, miR-126 overexpression expands specific progenitor cell subsets, which is reflected in an increased multi-lineage output during the first weeks after transplant.

SILAC Analysis Reveals PI3K/AKT Signaling is Target of miR-126
As the effect of miRNA modulation of gene expression may be stronger at the protein level and to provide another unbiased approach, we performed a quantitative analysis on the proteome of

K562 cells stably transduced with 126OE or CTRL vectors using the SILAC (Stable Isotope
Labeling of Amino acids in Culture) technique. We were able to reliably quantify 2,273 protein groups, and 510 of them showed a significant change (p<0.05) between 126OE and CTRL samples (Fig. S5a). We then interrogated this dataset using unsupervised Ingenuity Pathway Analysis (IPA) and found that the PI3K/AKT signaling pathway was among the most significantly affected canonical pathways upon 126OE (Fig. S5b,c), validating the bioinformatic analysis of the mRNA arrays. Taken together, our data pinpoints PI3K/AKT signaling as the foremost target of miR-126 in hematopoietic cells.

Mouse strains
B6.SJL-Ptprc a (CD45.1) and C57BL/6NTac-Ptprc b (CD45.2) mouse strains were purchased from Charles Rivers Laboratories (Milan, Italy) and maintained in specific-pathogen-free (SPF) conditions. CD45.1 and CD45.2 mice were also crossed to generate CD45.1-CD45.2 double positive mice to be used as recipients of competitive bone marrow transplants. All the procedures involving animals were designed and performed with the approval of the Animal Care and Use Committee of the Fondazione San Raffaele del Monte Tabor (IACUC #324) and communicated to the Ministry of Health and local authorities according to Italian law.

Lentiviral Constructs and Cord Blood Sample Preparation
Lentiviral vector platforms for ectopic miRNA expression and stable knockdown were described previously (Gentner et al., 2008;Amendola et al., 2009). For ectopic expression, the 717 bp genomic fragment containing human pri-mir-126 was cloned into the intron of LV.EF1.intron.LNGFR . In a second step, the EF1 promoter was substituted by the spleen focus forming/enhancer promoter (Gentner et al., 2008), and the LNGFR marker gene was replaced by the Orange Fluorescent Protein mOrange2 (OFP, (Shaner et al., 2008). For stable knockdown, 8 tandem copies of an imperfectly complementary miR-126 target sequence (5' gcattattactgatccacggtacga 3') were synthesized as described (Gentner et al., 2008) and cloned into the LV.SFFV.GFP or LV.SFFV.intron.OFP backbones.
Third generation lentiviral vector particles pseudotyped with VSV-G were generated as described (Follenzi et al., 2000;Guenechea et al., 2000). CB samples were obtained from normal full term deliveries after informed consent, according to procedures approved by the institutional review boards of the University Health Network and Trillium Hospital, or the San Raffaele Institute (TIGET01). Alternatively, CB cells were bought from commercial sources ("Poietics"

Colony forming cell assay
Human clonogenic assays were plated at day 3, 7 or 14 of culture in a methylcellulose-based medium (MethoCult H4434, StemCell Technologies). The following cell numbers were plated: day 3, 0.8x10 3 /ml; day 7, 2x10 3 /ml; day 14, 10x10 3 /ml. Two weeks after plating, colonies were counted under an inverted microscope and identified according to morphological criteria.
Mouse clonogenic assays were performed in M3434 Methocult (StemCell Technologies) by plating 1x10 3 /ml lineage depleted progenitors. Colonies were counted and identified according to morphological criteria after 1 week of culture, plucked and brought into single cell suspension and replated in fresh methylcellulose medium as a constant fraction of the resuspension volume roughly corresponding to 5x10 4 cells.

Cell sorting
After thawing, human lin-CB cells were resuspended at 10 7 cells / mL and stained with surface

Ki67 and Hoechst flow cytometry
Cells were stained for surface markers, washed and fixed using BD Cytofix buffer (Cat. #554655), washed and permeabilized with BD Perm 2 (Cat. # 347692), washed and stained with PE-or FITC-or PerCP-Cy5.5 conjugated Ki67 antibody (BD) and finally resuspended in BD Cytofix buffer with Hoechst at 1 µg/mL. The cells were then analyzed on a BD LSRII machine with UV laser.
Genomic DNA was extracted from cells after a minimum of 14 days in culture after LV transduction in order to get rid of non-integrated vector forms (Follenzi and Naldini, 2002). All reactions were carried in out in triplicate in an ABI Prism 7900HT (Applied Biosystems, Foster City, CA).
miRNA expression was analyzed as described (Gentner et al., 2008). Briefly, small RNAs were extracted using miRNeasy mini kit (Qiagen) and miRNA expression levels were determined by the Applied Biosystems Taqman® microRNA Assay system. Reactions were carried out in triplicate in an ABI Prism 7900HT (Applied Biosystems, Foster City, CA). miRNA expression was normalized to miR-16 or RNU48.
For gene expression analysis, total RNA was isolated from cells in culture using either the RNEasy mini or micro kit (Qiagen) according to the manufacturer's instructions. Retro transcription of total RNA was performed using Invitrogen Superscript III First Strand Synthesis TM System for RT-PCR, by loading a maximum of 500 ng of total RNA per reaction.
Taqman® gene expression analysis was performed using the following primer/probe sets

Western Blot and SILAC
Total cellular proteins were extracted with RIPA buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors: 1 mM PMSF, 10 mM NaF, 1 mM Na 3 VO 4 , CompleteMini™ and PhosStop ™ (Roche). Samples were resuspended in the lysis solution and incubated at 4°C for 30 min. Cell lysates were cleared by centrifugation at 10,000 × g for 10 min at 4°C, and the supernatants were collected and assayed for protein concentration using Lowry assay based method (DC, BioRad). 40-50 micrograms of proteins were run on SDS-PAGE under reducing conditions. For immunoblotting, proteins were transferred to nylon membranes (Hybond+, Amersham), incubated with the specific antibody followed by peroxidase-conjugated secondary antibodies (ECL Mouse IgG or ECL Rabbit IgG; GE Healthcare) and detected using chemiluminescent reagents (Luminata Forte, Millipore / ECL prime, Amersham) and exposure to autoradiography films.
For quantitative proteomics, K562 cells transduced with the 126OE vector or the CTRL vector were analyzed using Stable Isotope Labeling of Amino acids in Culture (SILAC) method (Ong et al., 2002). Briefly, cells were cultured in DMEM medium deficient in arginine and lysine (from Invitrogen) that was supplemented with stable isotope-encoded arginine and lysine (Sigma-Aldrich). For "heavy" labeling we used l-[ 13 C 6 , 15 N 4 ]arginine (Arg10) and l-[ 13 C 6 , 15 N 2 ]lysine (Lys8) and for the "light" condition l-[ 12 C 6 , 14 N 4 ]arginine (Arg0) and l-[ 12 C 6 , 14 N 2 ]lysine (Lys0) were used. In each SILAC condition, medium was supplemented with 10% dialyzed fetal calf serum (10KDa cutoff) and 1% (10 mg/ml) streptomycin/(10,000 units/ml) penicillin, and 1% lglutamine (200 mm in 0.85% NaCl; all from Invitrogen). General cell culture conditions were 37 °C, 5% CO 2 , and humidified atmosphere. After 2 weeks in culture (corresponding to > 6 cell doublings), cell cultures were mixed at an equal ratio and proteins were extracted, separated into 4 fractions (cytoplasm, nucleus, membrane and cytoskeleton, according to Qproteome fractionation kit, Qiagen) and run on SDS-PAGE. Single bands were cut from gel, reduced, alkylated and digested with trypsin as described (Shevchenko et al., 1996). The resulting peptide mixture was separated in multiple run of nanoscale C 18 reverse-phase liquid chromatography (Proxeon) coupled online to an LTQ-Orbitrap mass spectrometer (Thermo). Raw MS spectra processing and protein quantification was performed using freeware MaxQuant software (Cox, NatBt 2008) and statistical analysis on overrepresented pathways was conducted using Ingenuity Pathway Analysis (Ingenuity).

Analysis of Chimerism
Chimerism of congenic donor cells was expressed as percentage of cells expressing the fluorescence protein transduction marker (destabilized GFP (dGFP) or OFP). Only for the peripheral blood of mice carrying dGFP LVs, the chimerism was expressed as percentage of CD45.1 or CD45.2 cells, due to the sub-optimal expression of dGFP in some of the subpopulations of interest.

Microarray
RNA from transduced human CB cells was extracted using Trizol (Invitrogen) and gene expression assayed on HT-12_v4 microarrays (Illumina). Quantile normalization was performed and probes were filtered by detection p-value (0.1) (GeneSpring GX, Agilent). Next, to remove uninformative probes, those that did not exceed a threshold of 7.8 in all replicates of any one condition were eliminated, leaving 15812 probes for analysis.

Pathway and Network Analysis
The gene expression data were analyzed using GSEA (Subramanian et al., 2005) (Merico et al., 2010)) was generated using enriched gene-sets with a nominal pvalue <0.001, FDR<1% and the overlap coefficient set to 0.5. To identify gene-sets that may be significant due to variability in the CTRLs, GSEA analysis was repeated on 126KD vs. 126OE CTRLs (three CTRLs each, total six) and all nine possible permutations thereof. The most frequently appearing gene-sets observed in these comparisons were removed from the enrichment results (frequency greater than the median of the sum of the GSEA nominal enrichment scores (NES) for each gene-set across all permutations).
The list was compared to enriched gene-sets in the enrichment map and overlap was scored using Fisher's Exact Test p-value. Overlaps with p-value smaller than 0.05 were visualized.

Flow cytometry
Routine flow cytometry was performed using BD LSRII and BD Canto cytometers. Data was analyzed with FlowJo software (Tree Star, Inc.) or FCS Express software (DeNovo Software).
The following antibodies against human surface markers were used: In vivo nucleoside incorporation assay In vivo proliferation assays were performed at 3 weeks after bone marrow transplant, using 5ethynyl-2'deoxyuridine (EdU, Invitrogen), a nucleoside analogue to thymidine used as an alternative to BrdU. EdU was dissolved in sterile 1x PBS at a concentration of 10 mg/ml. Mice received two pulses of EdU (i.p.), 100µg each, 24 and 12 hours before sacrifice. BM was harvested and HSPCs were purified by pools of 2 or 3 mice (Lineage cell depletion kit, Miltenyi Biotec). Enriched HSPCs were processed according to manufacturer's instructions (Click-iT® EdU Flow Cytometry Assay Kit, Invitrogen), and percentages of EdU incorporation into HSPC subsets were measured by FACS.

Phosphoflow
Phosphoflow cytometry was performed as published elsewhere (Krutzik et al., 2011). Briefly, CD34+ cells cultured in TSF6 (see Viral transduction of cord blood and in vitro expansion) were washed with fresh StemSpan medium and pelleted to eliminate conditioned medium. After 2 washes, cells were starved in cytokine-free StemSpan medium at 37°C for 3 hours. Then, cells were stimulated with TSF6 for 5, 10 or 15 minutes prior to fixation. As a negative control, cells were administered 100 nM Wortmannin (CST) 30 minutes before stimulation with TSF6. Cells were immediately fixed with paraformaldehyde (final concentration: 1.6%) for 10 minutes at room temperature. Cells were then centrifuged, washed once with PBS 1 % BSA to remove residual PFA and permeabilized with ice-cold Perm buffer III (BD Phosflow for 30 min at 4°C followed by 2 washes in order to remove traces of methanol). Antibody staining was then performed. The GFP fluorescence was augmented by antibody staining (FITC-conjugated polyclonal goat anti-GFP, Abcam ab6662). Optionally, surface staining for CD133 and CD34 was performed. Phospho-Akt Ser473 was detected by a monoclonal antibody (clone D9E; Cell Signaling) conjugated to Alexafluor 647 (#4075). Phospho-GSK3 (Ser9) was detected by a rabbit monoclonal unconjugated (clone D85E12, Cell Signaling #5558) combined with an Alexa 647 conjugated secondary antibody. Staining was performed by incubating permeabilized cells with the phospho-specific antibody for 30 minutes on ice and at dark, diluted in PBS 1% BSA at a final concentration of 1:50. After incubation, cell were washed in PBS 1% BSA and then analyzed by flow cytometry. Since the efficiency of 126KD is directly dependent on the amount of miR-126 targets expressed per cell which varies according to the transduction level achieved by the 126KD vector, different pre-gates on GFP or OFP expression were set to stratify the cells into poorly vs. highly transduced cells. GFP/OFP gates were defined by dividing the spectrum of fluorescence marker expression achieved by each vector into quartiles and by choosing the 10% of cells with highest fluorescence marker expression. Within these pre-gates, the mean fluorescence intensity (arithmetic mean) of Phospho-Akt (Ser473) or Phospho-GSK3β (Ser9) was determined for CTRL, 126KD or 126OE vector transduced cells. As a negative control, the samples were pretreated with Wortmannin. Statistical differences between 126KD LV and CTRL LV transduced cells were revealed by a paired t-test.