Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering / 대한해부학회지

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

The Effect of C/EBPalpha or C/EBPepsilon on 32Dcl3 Murine Myeloblasts and #1111 Acute Promyelocytic Leukemic Cells / 대한진단검사의학회지

BACKGROUND: The pathogenesis of acute myeloid leukemia (AML) is complicated by DNA damage, balanced or unbalanced translocation, deletion, inversion, abnormal transcription factors, receptors, and others. The CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBP epsilon (C/EBPepsilon), one of transcription factors, play important roles in normal granulopoiesis. We wished to assess whether increasing the activity of either C/EBPalpha or C/EBPepsilon could suppress the leukemic myeloblasts. METHODS: To make retrovirus, BOSC23 cells were transfected with retroviral constructs; mouse stem cell retrovirus-internal ribosomal entry site-green fluorescent protein (MIG), MIG-C/EBP alpha-estrogen receptor (ER) and MIG-C/EBP epsilon-ER. 32Dcl3 murine myeloblastic (32Dcl3) cells or #1111 acute promyelocytic leukemic (#1111 APL, #1111) cells were transduced with each retrovirus. Growth rate and differential cell count were examined, and granulocytic surface markers of Gr-1 and Mac-1 were checked. Transduced #1111 cells were injected into 20 sublethally irradiated (4.5 Gy) mice; at day 14, 4 groups of 5 mice each were input into subcutaneous tissue with placebo, 4-hydroxytamoxifen (4HT), all trans retinoic acid (ATRA), or 4HT & ATRA pellets; survival times were analysed when they died. RESULTS: The number of GFP (+) transduced 32Dcl3 cells with MIG (control group) at days 2, 4, and 6 were 684976, 1975965, and 2808244; 32Dcl3 cells with MIG-C/EBP alpha-ER were 77354, 53180, and 39460; and 32Dcl3 cell with MIG-C/EBP epsilon-ER were 328384, 698424, and 974850, respectively. The control group didn't express both Gr-1 and Mac-1, but C/EBP alpha expressed 56.1%, 55.6% and C/EBPepsilon expressed 31.3% and 32.6%, respectively. The differential counts of immature, intermediate, and mature forms in control group were 90.0%, 6.0%, and 4.0%; C/EBP 4.3%, 33.7%, and 62.0%; C/EBPepsilon 41.0%, 48.3%, and 10.7%, respectively. The mean survival time of transduced #1111 cells with MIG-C/EBP alpha-ER injected mice was 30.5 days in placebo group, 41.8 days in 4HT (C/EBP ) group, 69.0 days in ATRA group, and 97.8 days in 4HT (C/EBP ) & ATRA group. In case of MIG-C/EBP epsilon-ER, the survival time was 26.4 days in placebo group, 33.0 days in 4HT (C/EBP ) group, 49.6 days in ATRA group, and 52.5 days in 4HT (C/EBP ) & ATRA group. CONCLUSIONS: Both C/EBP and C/EBP suppressed cell growth and differentiation of 32Dcl3 cells, and they also suppressed cell growth of #1111 cells. The ATRA was more effective than C/EBP in APL, and C/EBP and ATRA had synergistic effects in APL. The growth arrest and differentiated action of C/EBPalpha was more powerful than that of C/EBPepsilon.