Down Regulation of GATA-2 and the Inversely-Correlated Expression of Adipogenic Specific Gene-PPARγ in Mesenchymal Stem Cells from Patients with Aplastic Anemia.

(Introduction) Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells resulting in peripheral pancytopenia and fatty marrow replacement. Although immune-mediated suppression of hematopoiesis is considered to play an important role in most patients with AA, the mechanism of fatty marrow replacement remains unclear. Transcription factor GATA-2 is known to play important roles in hematopoiesis. Gene Chip analysis has implicated the decreased expression of GATA-2 in AA CD34 cells, a finding confirmed by quantitative real-time PCR analysis. In a mouse stromal preadipocyte cell line, the suppression of GATA-2 expression accelerated the differentiation of stromal preadipocytes. In order to explore the impact of GATA-2 expression on adipogenesis in AA, the expression of GATA-2, the adipogenic specific gene adipsin, CCAAT/enhancer binding protein alpha (C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ) was examined in mesenchymal stem cells (MSCs) derived from bone marrow from AA patients and normal individuals.

(Patients and methods) Thirty-four AA patients and 15 healthy individuals were included in this study. The median patient age was 10 years, ranging from 2 to 21 years. Twelve patients were studied before immunosuppressive therapy (IST) or bone marrow transplantation. Twenty-two patients were studied after treatment, of whom 11 responded to IST and 11 did not respond. Mononuclear cells were separated from bone marrow aspirates and 1x10⁷ cells used for MSCs culture. MesenCult (StemCell Technologies, USA) was used to culture and expand the MSCs in vitro, after which the cells retain their ability to differentiate along various lineages upon the addition of the appropriate stimulatory supplements. Total RNA was extracted from the third passage of MSCs and synthesis of cDNA was performed using the Thermoscript RT-PCR system. Quantitative real-time PCR was conducted using Taqman primers and probes. The K562 cell line for GATA-2, the EOL1 cell line for adipsin and the MOLM-13 cell line for C/EBPα and PPARγ were used as controls.

(Results) GATA-2 expression by MSCs from patients with AA at diagnosis was significantly decreased compared with normal individuals (p=0.031). However, one of the adipogenic specific genes, PPARγ, was significantly increased in MSCs in AA at diagnosis (p=0.017). The expression of adipsin and C/EBPα in MSCs did not differ significantly between AA patients and healthy volunteers. Notably, in the 22 AA patients who received IST, the expression of GATA-2 and PPARγ did not significantly decrease if the patient had responded and hematopoiesis had improved (n=11). However, after the IST the 11 non-responders still had significantly lower GATA-2 and higher PPARγ expression compared to normal individuals.

(Conclusion) The mechanism of fatty replacement in the bone marrow in AA may be explained by the down regulation of GATA-2 and the inversely-correlated expression of PPARγ in MSCs. The decreased expression of GATA-2 may be responsible for the pathogenesis and development of the clinical features of this disease.