Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

Abstract

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73⁺ CD105⁺ CD271⁺ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

Figure 1

Isolation of the rare BMSC population from primary bone marrow samples. (A) Workflow: Mononuclear cells (MNCs) from fresh bone marrow (BM) aspirates of patients newly diagnosed with MDS or AML as well as healthy donors were obtained by density gradient centrifugation and stored in liquid nitrogen until use. For BMSC isolation, BM-MNCs were thawed and subsequently sorted by FACS. (B–D) Gating strategy for BMSC isolation. After forward/sideward scatter gating, doublet and dead cell exclusion (using propidium iodide staining), living cells were sorted on CD45^-, lineage (CD235a/CD31)^-, CD271⁺, CD73⁺ and CD105⁺ surface marker expression for purification of BMSCs. Representative FACS plots are shown for healthy donor (B), MDS (C), and AML (D) samples. (E) Uncultivated BM-MNC from healthy donors (n = 21), MDS (n = 22) and AML (n = 30) samples were analyzed on CD45−/CD31−/CD235a−/CD271+/CD73+/CD105+ expression by FACS, and statistical frequencies of the subpopulations were calculated. Event counts of the subpopulations were normalized on 5 × 10⁴ total event counts per sample. Medians are indicated in black.

Figure 2

Formation of CFU-F-derived colonies is only found in a specific population subset and is strongly reduced in MDS and AML samples. (A) Experimental design. The CD45⁻/CD31⁻/CD235a⁻/CD271⁺/CD73⁺/CD105⁺ (+/+) and CD45−/CD31−/CD235a−/CD271⁺/CD73−/CD105− (−/−) subpopulations were simultaneously sorted from BM-MNC from healthy donors, MDS, and AML samples by FACS. Sorted subpopulations were seeded separately to assess their potential to form CFU-F-derived colonies, followed by re-analysis of their surface marker expression profile after two passages by FACS. (B) Colony-forming efficiency (CFE, normalized on 1 × 10⁴ input cells) in the +/+ and −/− subpopulations of healthy donors (n = 6), MDS (n = 7), and AML (n = 1) samples. Medians are indicated in black. (C) CFE (normalized on 1 × 10⁴ input cells) in the +/+ subpopulation of healthy donors (n = 16), MDS (n = 14), and AML (n = 20) samples. Medians are indicated in black. (D) Kaplan–Meier curve for replating efficiency (%) of +/+ sorted samples after initial seeding (0), passage 1, and passage 2. Healthy donors, n = 6; MDS, n = 6; AML, n = 9. (E) Representative light microscopy pictures showing the morphology of +/+ sorted cells from healthy donors, MDS, and AML samples at day 2 after passage 1. Cells were sorted, seeded and expanded at the same time and conditions. Scale bars = 100 µm. (F) Re-analysis of surface marker expression of +/+ sorted cells by FACS after passage 2. Representative histograms are shown for a healthy donor sample. Grey filled lines indicate IgG controls, red lines the respective antibodies.

Figure 3

Significantly increased in vitro adipogenic differentiation potential in BMSCs from MDS and AML samples. (A) Experimental setup. CD45−/lin(CD31/CD235a)−/CD271⁺ sorted MSC from healthy donors, MDS, and AML samples were seeded and CFU-F-derived colonies were expanded until confluent. Osteogenic and adipogenic differentiation were induced in vitro for 21 days. Osteoblasts were detected by Alizarin Red staining of calcium deposits. Adipocytes were detected by Oil Red staining of fatty vacuoles. Stained samples were categorized according to a staining intensity score ranging from 0 (= no staining) up to 4 (= maximum staining). (B) Quantification of in vitro osteogenic differentiation potential of sorted BMSCs from healthy donors (n = 6), MDS (n = 6), and AML (n = 6) patient samples. Medians are indicated in black. (C) Representative images of Alizarin Red staining of osteogenic differentiated healthy donor, MDS, and AML samples (top row) and their respective undifferentiated controls (bottom row). Scale bars = 100 µm. (D) Quantification of in vitro adipogenic differentiation potential of sorted BMSCs from healthy donors (n = 7), MDS (n = 5), and AML (n = 6) patient samples. Medians are indicated in black. (E) Representative images of Oil Red staining of adipogenic differentiated healthy donor, MDS, and AML samples (top row) and their respective undifferentiated controls (bottom row). Scale bars = 100 µm.

Figure 4

Expression of the adipogenic inhibitor Delta-like 1 (DLK1) is strongly reduced in MDS and AML BMSC. (A) Experimental setup: CD45−/lin(CD31/CD235a)−/CD271+/CD73+/CD105+ sorted +/+ MSC from healthy donors, AML and MDS samples were seeded and CFU-F-derived colonies were expanded and cultured BMSC harvested for gene and protein expression analysis. (B) Gene expression analysis by qRT-PCR of the orphan Notch/Delta/Serrate family-ligand DLK1, the Notch pathway receptors NOTCH1 and NOTCH3, the osteogenic cell fate stimulator WNT10B, the mature adipogenic differentiation markers PPARγ and LPL, and the mature osteogenic differentiation markers RUNX2 and SPP1 in cultured BMSCs from healthy donors (DLK1: n = 12, NOTCH1, NOTCH3, WNT10B, and PPARG: n = 11, each, LPL, and RUNX2: n = 8, each, SPP1: n = 10), MDS (DLK1: n = 13, NOTCH1 and NOTCH3: n = 12, each, PPARγ: n = 9, LPL, WNT10B, RUNX2, and SPP1: n = 8, each), and AML (DLK1, NOTCH1, NOTCH3, and WNT10B: n = 8, each, PPARγ, LPL, RUNX2, and SPP1: n = 7, each) samples. Data are shown as relative mRNA expression of the respective target gene to EIF3 mRNA expression using the ΔΔCt method with medians indicated in black. (C–E) Analysis of DLK1 protein expression in sorted BMSCs from healthy donors, MDS, and AML samples via immunoblotting. Exemplary immunoblots are shown in (C) with β-ACTIN as housekeeping control. Numbers indicate individual patient samples. H = healthy donors. Quantification of immunoblotting in (D,E) is shown as relative protein expression of DLK1 normalized on β-ACTIN expression, with 1 = 100% β-ACTIN expression. Medians are indicated in black. (D) Healthy donors n = 7, MDS n = 4. (E) Healthy donors n = 6, AML n = 4.