Constitutively active beta-catenin confers multilineage differentiation potential on lymphoid and myeloid progenitors

Abstract

Beta-catenin-mediated Wnt signaling may contribute to the self-renewal of hematopoietic stem cells and proliferation in some malignancies. We now show that expression of constitutively active beta-catenin in normal lymphoid or myeloid progenitors generated uncommitted cells with multilineage differentiation potential. Inappropriate gene expression occurred in cells destined to produce either cell type and caused corresponding changes in their characteristics. For example, forced activation of beta-catenin quickly increased C/EBPalpha while reducing EBF and Pax-5 in lymphoid progenitors that then generated myeloid cells. Inversely, EBF dramatically increased in transduced myeloid progenitors and lymphocytes were produced. The results indicate that ectopic activation of beta-catenin destabilizes lineage fate decisions and confers some, but not all, stem cell properties on committed progenitors.

Figure 1. Expression of constitutive stable β-catenin in lymphoid progenitors confers myeloid lineage differentiation potential

(A) Transduced GFP⁺ lymphoid progenitors with either control vector or stable β-catenin were sorted and co-cultured for 14 days on OP9 stromal cells with SCF, FL and IL-7. Flow cytometry was then used to detect various myeloid and B lineage lymphoid cells. (B) Mac-1⁺ cells were sorted after OP9 co-culture and a May-Grunwald-Geimsa stained cytospin preparation is shown. (C) After the first 7 days, and at 7 day intervals, cells were harvested and counted, and then 10,000 were re-plated on fresh OP9 stromal cells. (D) CD11b/Mac-1⁺ cells with or without the CD11c dendritic marker were recovered from 14 day cultures and purified genomic DNA was evaluated for Ig_H gene rearrangements. Freshly sorted CD19⁺ cells and Lin⁻ c-Kit^Lo Sca-1^Lo IL-7Rα⁺ lymphoid progenitors from bone marrow were used as positive controls and compared to CD11b/Mac-1⁺ cells generated from HSC fraction in culture.

Figure 2. Expression of constitutive stable β-catenin in lymphoid progenitors confers multi-lineage differentiation potential

(A) CD19⁻ Mac-1⁻ cells in 14 day primary cultures initiated with stable β-catenin were sorted and tested for differentiation potential. Cells sub-cultured onto OP9 with FL and IL-7 generated both CD19⁺ B and CD11b/Mac-1⁺ lineage cells (left two panels). CD3ε T lineage cells emerged within 3 weeks onto OP9 stromal cells transduced with the Delta-like1 Notch ligand (OP9-DL1) (right two panels). (B) Lymphoid progenitors transduced with control vector or β-catenin were co-cultured on OP9 stromal cells in the presence of SCF, FL and IL-7. After 14 days, 1x10⁶ cells were injected into sublethally irradiated NOD/SCID mice. After 5 weeks, cells were isolated from spleens before analysis by flow cytometry.

Figure 3. Ectopic activation of β-catenin in myeloid progenitors confers multi-lineage differentiation potential

(A) GFP⁺ myeloid progenitors transduced with control vector or stable β-catenin were sorted and co-cultured on OP9 stromal cells in the presence of SCF, FL and IL-7. B (CD19) or myeloid (Mac-1) lineage differentiation was assessed by flow cytometry at 10 days. May-Grunwald-Geimsa staining of sorted CD19⁺ cells is shown. (B) After the first 10 days, and at 10 day intervals, cells were harvested and counted, and then 10,000 were re-plated on fresh OP9 stromal cells. (C) CD19⁻ Mac-1⁻cells derived from stable β-catenin expressing myeloid progenitors were sorted and allowed to differentiate to CD19⁺ and Mac-1⁺ cells on OP9 stromal cells in the presence of FL and IL-7 during an additional 10 days (left panel). Sorted CD19⁻ Mac-1⁻cells also gave rise to αβT and γδT cells when held for 3 weeks on OP9-DL1 stromal cells in the presence of FL and IL-7 (right three panels). (D) Myeloid progenitors transduced with control vector or β-catenin were co-cultured on OP9 stromal cells for 10 days in the presence of SCF, FL and IL-7 and 1 x10⁶ cells were injected into sublethally irradiated NOD/SCID mice. After 5 weeks, cells were isolated from spleen and analyzed by flow cytometry.

Figure 4. One subset of myeloid progenitors efficiently gives rise to B lineage cells following β-catenin activation

Lin⁻ c-Kit^Hi Sca-1⁻ CD34⁺ FcγRII/III^Lo CMP and Lin⁻ c-Kit^Hi Sca-1⁻ CD34⁺ FcγRII/III^Hi GMP were sorted and transduced with either control vector or stable β-catenin. GFP⁺ cells were then isolated and co-cultured on OP9 stromal cells in the presence of SCF, FL and IL-7 for 10 days. The bar graphs indicate yields, i.e., numbers of CD19⁺ B, CD11b/Mac-1⁺ myeloid lineage or CD19⁻ CD11b/Mac-1⁻ undifferentiated cells recovered per input cell.

Figure 5. Altered lineage fates of lymphoid and myeloid progenitors resulting from activation of β-catenin

Single sorted GFP⁺ cells from a HSC enriched fraction, lymphoid progenitors or myeloid progenitors transduced with control vector or stable β-catenin were cultured on OP9 stromal cells in 96-well plates for 12 days in the presence of SCF, FL and IL-7. Positive readout wells (more than 40 cells) were determined by microscopic observation and phenotypes were determined by flow cytometry (representative examples shown on top row). The frequencies of wells with each of these differentiation patterns are shown along with total numbers of clones observed.

Figure 6. Stable β-catenin alters lineage-associated gene patterns

(A, B) Progenitors of the indicated types were transduced with control vector or stable β-catenin for 48 hours before sorting for GFP⁺ cells. Freshly isolated lymphoid progenitors and myeloid progenitors were used as controls. Semi-quantitative RT-PCR was carried out to amplify transcripts for the indicated genes in each population. The bar graphs represent the results of [α ³²P] dCTP incorporation on the linear parts of PCR amplification curves normalized relative to β-actin control values as detailed in Materials and Methods.