Wnt5a Does Not Support Hematopoiesis in Stroma-Free, Serum-Free Cultures

<div><p>Previously we reported that Wnt5a is highly expressed in the murine urogenital ridge-derived UG26-1B6 cells but not embryonic liver-derived EL08-1D2 cells. Mouse long-term repopulating hematopoietic stem cells (LTR-HSC) were maintained in non-contact UG26-1B6 cultures but not EL08-1D2 non-contact cultures, unless Wnt5a was also added to the cultures, suggesting a role for Wnt5a in the <em>in vitro</em> maintenance of LTR-HSC. Here, we investigated if the effect of Wnt5a on adult LTR-HSC activity is HSC-autonomous. To test the effect of Wnt5a on maintenance of LTR-HSC, we performed limiting dilution competitive transplantation assays of murine Lin-Sca1⁺ c-kit⁺ (LSK) cells cultured for 5 days with TPO and SCF with and without Wnt5a. The effect of Wnt5a on the generation of colony forming units (CFU) and the homing ability of LSK progeny was also tested. No effects were found of Wnt5a on total cell expansion, the number of CFU, or homing ability of day 5 LSK progeny. Furthermore, addition of Wnt5a did not improve, but may have impeded maintenance of LTR-HSC. In conclusion, our data indicate that Wnt5a does not enhance the maintenance and expansion of adult murine LTR-HSCs or committed progenitors cultured <em>in vitro</em> in serum- and stroma-free conditions.</p> </div

Multilineage engraftment analysis in secondary recipients of LSK progeny cells cultured with and without Wnt5a.

<p>One million BM cells from positively repopulated (≥1% CD45.1 multilineage engraftment) primary recipients of LSK cell progeny cultured with TS or TSW were grafted in secondary CD45.2 recipients. Multilineage CD45.1 engraftment was evaluated after 4 months (4 M) in the peripheral blood (PB) (A) Numbers above the graph represent the number of positively reconstituted (≥1% CD45.1 multilineage engraftment) mice/total number mice injected per group. The graph represents mean with SEM of percent chimerism per group. (B) Competitive repopulation unit (CRU) frequency in primary and secondary recipients calculated using ELDA software (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053669#pone.0053669-Hu1" target="_blank">[18]</a>); * = <i>p≤0.05</i>.</p

Multilineage engraftment analysis in primary recipients of LSK progeny cells cultured with and without Wnt5a.

<p>CD45.2 recipients were transplanted with progeny of 50, 100 or 200 CD45.1 LSK cells cultured for 5 days with TPO and SCF alone (TS) or with Wnt5a (TSW) combined with 10⁵ CD45.2 bone marrow (BM) cells. Multilineage CD45.1 engraftment was evaluated after 4 months (4M) in the peripheral blood (PB) (A) Shown are the percentages of CD45.1⁺ cells in PB at 4M posttransplantation in each primary positively repopulated recipient (≥1% overall donor-derived cells contributing to multilineage engraftment) represented by rhombus in different conditions indicated in the figure. (B) Numbers above the graph represent the number of positively reconstituted (≥1% CD45.1 multilineage engraftment) mice/total number injected mice per group. The graph represents mean with SEM of donor-derived engraftment in primary positively repopulated recipients per group. * = <i>p≤0.05.</i> (C, D, E) Mean with SEM donor myeloid (CD11b⁺cells), T-lymphoid (CD4/CD8⁺ cells) and B-lymphoid (B220⁺ cells) chimerism per group. * = <i>p≤0.05</i>.</p

<i>In vivo</i> homing of LSK cell progeny cultured <i>ex vivo</i> with and without Wnt5a.

<p><i>In vivo</i> homing of LSK cell progeny cultured for 5 days with TPO and SCF alone (TS) or with Wnt5a (TSW) was measured by scoring CFU-C in 6×10⁵ BM cells, harvested 16 hrs after infusion of 25,000 LSK cell progeny into lethally irradiated mice. BM cells harvested from nonirradiated mice served in this homing assay as a positive control readout for CFU-C (defined as control in the graph); PBS = BM from mice that were lethally irradiated and injected i.v. with PBS served as negative control; LSK TS 5d = BM from mice that were lethally irradiated and injected with 5days TS-treated LSK progeny cells; LSK TSW 5d = BM from mice that were lethally irradiated and injected with 5 days TSW-treated LSK progeny cells. The data represent here the mean of 3 experiments with SEM of CFU-C frequency in the BM.</p

Gene expression primers.

<p>Gene expression primers.</p

EZH2 regulation and fetal hepatocytes differentiation.

<p><b>A.</b> Upper part, directed differentiation of hPCS-<i>i</i>EZH2 toward fetal hepatocytes (FH_d16) with addition of 5μg/ml doxycycline (doxy) in the first 8 days of differentiation (red arrow). Bottom part, relative gene expression of the <i>EZH2</i> transgene (Exogenous <i>EZH2</i>) and both endogenous and exogenous EZH2 (Total <i>EZH2</i>) at endo_d4, HP_d8 and FH_d16 in untreated (-) and EZH2 doxy induced cells (+). Relative gene expression to GAPDH and same scale bar. Data as mean ± SEM of n ≥ 3 IEs. <b>B.</b> Western blot for EZH2 and GAPDH in untreated (-) and EZH2 doxy induced cells (+) at hPSC, endo_d4, HP_d8 and FH_d16. Positive control: HEK293T cells transiently transfected with pLVX-IRES-Hygro-hEZH2. The molecular weight size marker was cropped from the gel. Signals were quantified and indicated as % to loading control. <b>C.</b> Relative expression (to <i>GAPDH</i>) of hepatic markers <i>HNF1A</i>, <i>HNF3B</i>, <i>HNF6</i>, <i>GSTp</i> and transcription factors <i>CAR</i> and <i>CEBPA</i> in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05 and ** p < 0.01. <b>D.</b> Relative gene expression (to <i>GAPDH</i>) of hepatic marker genes <i>AFP</i>, <i>ALBUMIN and AAT</i> in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05, ** p < 0.01. <b>E.</b> Left part, relative gene expression (to <i>GAPDH</i>) of hepatic transcription factor HNF4A in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Right part, representative immunofluorescence images for HNF4A (red signal) on day 8 for cells treated without (untreated) or with doxy (doxy-treated). Nuclei are staining with DAPI (blue). On the right, more than 80% of HNF4A positive cells on HP_d8 EZH2 doxy induced cells were counted. Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05. <b>F.</b> Immunofluorescence staining for ALBUMIN (red signal) at HP (day8) and FH (d16) in untreated (-) and EZH2 induced cells (+) (left part). Nuclei are staining with DAPI (blue). Data as representative images of n = 2 IES. <b>G.</b> ELISA for ALBUMIN secretion on FH_d16 of the hepatocyte differentiation protocol. EZH2 doxy induced cells (+) secrete significant amounts of albumin compared to untreated (-) cells. Data as mean ± SEM of n ≥ 3 IEs. ** p < 0.01. <b>H.</b> Immunofluorescence staining for AAT (red signal) at HP (day 8) and FH (day 16) showed abundant expression of the hepatocyte protein in doxy-treated cells compared to the untreated (left part). Nuclei are stained with DAPI (blue). Data as representative images of n = 2 IES. <b>I.</b> Intracellular flow cytometry analysis for AAT demonstrated that more then around 60% of EZH2 doxy induced cells (+) progeny were positive for AAT. Results represent the mean of three independent experiments ± SEM. ** p < 0.01.</p

EZH2 expression during hepatocyte differentiation.

<p><b>A.</b> Upper part, differentiation protocol of hPSCs into hepatocytes-like cells. hESC were harvested and plated at ±7 x 10⁴ cell/cm² and differentiated with a combination of growth factors: 50ng/ml of Activin-A and Wnt3a for the first 2 days and 50ng/ml Activin-A until day 4, followed by BMP4 50 ng/ml from day 4 until day 8, FGF1 50 ng/ml from day 8 until day 12, and HGF 20 ng/ml from day 12 until day 16. Differentiated progeny of the hESC line were harvested on day 4 (endo_d4, definitive endoderm cells), on day 8 (HP_d8, hepatoblast progenitor) and on day 16 (FH_d16, fetal hepatocyte). Bottom part, transcript expression levels of <i>EZH2</i> and <i>RING1B</i> in hPSCs, endo, HP and FH stages. <i>GAPDH</i> was used as a control. Data as mean ± SEM of n ≥ 3 IEs. *** p < 0.001 by Student’s t test. <b>B.</b> Western blot analysis of EZH2, RING1B and GAPDH on hPSCs, endo, HP and FH stages. GAPDH was used as loading control. HEK293T transiently transfected with pLVX-IRES-Hygro-hEZH2 cells were used as positive control. Signals were quantified and indicated as % to loading control. <b>C.</b> Schematic representation of the <i>EZH2</i> regulatory region. Approximately 270bp (arrow in front of the TTS) in a CpG island were bisulfite sequenced in undifferentiated hPSC, HP and FH. Percentages of the results of bisulfite sequencing of the regions are at right side of the figure for each stage.</p

miRNAs expression primers.

<p>miRNAs expression primers.</p

miRNA and EZH2 mRNA degradation.

<p><b>A.</b> Relative gene expression (to <i>GAPDH</i>) of endogenous and exogenous <i>EZH2</i> (Total <i>EZH2</i>) daily during the transition from endoderm stage (day 4, endo_d4) to hepatoblast progenitor (day 8, HP_d8) in untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n = 3 IEs. <b>B.</b> Relative expression of miR-101, miR-138, miR-214 and miR-124 during hepatocytes differentiation from hPSC-<i>i</i>EZH2 cell line doxy induced the first 8 days of differentiation. Relative gene expression to U6. Data as mean ± SEM of n = 3 IEs. <b>C.</b> Name, functions and reference papers of the miRNAs analyzed.</p

hESC-inducible EZH2 (hPSC- <i>i</i>EZH2) cell line and definitive endoderm formation.

<p><b>A.</b> The original master cell line flanked by heterotypic FRT sequences and resulting RMCE line (Recombinase-Mediated Cassette Exchange, hPSC-<i>i</i>EZH2) are depicted. RMCE donor vector <i>pZ</i>:<i>F3-P TetOn hEZH2</i> for inducible expression was introduced by flippase (additional details in the Supplemental Information). <b>B.</b> Expression of pluripotency markers (TRA-1-60 and OCT4) of a representative hPSC-<i>i</i>EZH2 clone and wild-type hPSCs by immunocytochemistry. Right: isotype control of TRA-1-60 and OCT4. <b>C.</b> Upper part, directed differentiation of hPCS-<i>i</i>EZH2 toward endoderm (endo_d4) with addition of 5μg/ml doxycycline (doxy) in the first 4 days of differentiation (red arrow). Bottom part, protein analysis of untreated and doxy treated EZH2 induced cells (doxy -/+) using EZH2, GATA4 and GAPDH antibodies. GAPDH was used as loading control. Signals were quantified and indicated as % to loading control. <b>D.</b> Double extracellular staining for CXCR4 (PE channel) and cKIT (APC channel) measured by FACS in untreated and doxy treated EZH2 induced cells (doxy-treated) at definitive endoderm stage on day 4. Right: the percentage of cells co-expressing CXCR4 and cKIT. Data as mean ± SEM of n ≥ 3 IEs. ** p < 0.01 by Student’s t test. <b>E.</b> Representative immunofluorescence images for SOX17 (red signal) in untreated and EZH2 induced cells (doxy-treated) at day 4 of differentiation. EZH2 induced cells showed significantly high levels of SOX17 expression. Around 58% of positive cells were counted in EZH2 doxy induced cells, right part. Nuclei are staining with DAPI (blue). Data as mean ± SEM of n ≥ 3 IEs. *** p < 0.001 by Student’s t test. <b>F.</b> mRNA expression profile of the endoderm marker <i>SOX17</i> in untreated (-) and EZH2 doxy induced cells (+) at endo_d4. <b>G.</b> mRNA expression profile of untreated (-) and EZH2 doxy induced cells (+) at endo_d4 and HP_d8 of endoderm markers <i>FOXA2</i>, <i>EOMES</i>, <i>MIXL1</i>, <i>GOOSECOID</i> and <i>CXCR4</i>. Relative gene expression to GAPDH. Data as mean ± SEM of n ≥ 3 IEs.</p