Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia

Abstract

Aplastic anemia (AA) is generally considered as an immune-mediated bone marrow failure syndrome with defective hematopoietic stem cells (HSCs) and marrow microenvironment. Previous studies have demonstrated the defective HSCs and aberrant T cellular-immunity in AA using a microarray approach. However, little is known about the overall specialty of bone marrow mesenchymal stem cells (BM-MSCs). In the present study, we comprehensively compared the biological features and gene expression profile of BM-MSCs between AA patients and healthy volunteers. In comparison with healthy controls, BM-MSCs from AA patients showed aberrant morphology, decreased proliferation and clonogenic potential and increased apoptosis. BM-MSCs from AA patients were susceptible to be induced to differentiate into adipocytes but more difficult to differentiate into osteoblasts. Consistent with abnormal biological features, a large number of genes implicated in cell cycle, cell division, proliferation, chemotaxis and hematopoietic cell lineage showed markedly decreased expression in BM-MSCs from AA patients. Conversely, more related genes with apoptosis, adipogenesis and immune response showed increased expression in BM-MSCs from AA patients. The gene expression profile of BM-MSCs further confirmed the abnormal biological properties and provided significant evidence for the possible mechanism of the destruction of the bone marrow microenvironment in AA.

Figure 1. The representative morphology and multiple differentiation capacity of BM-MSCs from AA patients and healthy controls.

The morphology of BM-MSCs was shown after staining with β-tubulin (A). The adipogenic differentiation capacity of BM-MSCs detected by un-staining (B) and positive staining of Oil Red O (C). The osteogenic differentiation capacity of BM-MSCs detected by positive staining of Allizarin Red (D), von Kossa (E) and ALP (F).

Figure 2. The proliferation and CFU-F capacity of BM-MSCs.

(A) The proliferation rate of BM-MSCs were detected using BrdU-ELISA method after different incubation times. *P<0.05, ***P<0.001. (B) The clonogenic capacity was assessed by colony-forming unit-fibroblast (CFU-F) assay. Data represented mean ± SD (n = 10).

Figure 3. The apoptosis rate of BM-MSCs from AA patients and healthy controls.

The apoptotic rate of BM-MSCs was assessed with Annexin V Apoptosis Detection Kit (A). The representative dotplots of apoptotic rate of BM-MSCs from healthy controls (B) and AA patients (C). Data represented mean ± SD (n = 10).

Figure 4. Global view of gene expression profile of BM-MSCs.

Gene expression profile of BM-MSCs was determined using Affymetrix oligoarrays. (A) The scatter plot for two biological replicates. (B) The comparison of cluster data between AA patients and healthy controls. (C) Validation of GeneChip results by quantitative real-time PCR. Data were relative to the amount of GAPDH.

Figure 5. Gene expression patterns of BM-MSCs from AA patients in key biological signal pathways.

Gene expression patterns were grouped and displayed in the following categories: cell cycle, cell division, cell proliferation, apoptosis, chemotaxis, immune response, adipogenesis, and hematopoietic cell lineage. Relative genes expression were analyzed and compared between AA patients and healthy controls. Genes differentially expressed in BM-MSCs from AA patients were identified with at least a 2.0-fold change with respect to healthy control pools. The up-regulated genes are shown in red while down-regulated genes are shown in green.