Adipose Derived Stem Cells Affect miR-145 and p53 Expressions of Co-Cultured Hematopoietic Stem Cells

Abstract

Objectives: Umbilical cord blood is used for transplantation purposes in regenerative medicine of hematological disorders. MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as cancer development and incidence. There is a new relation between p53 (tumor suppressor gene) and miR-145 (suppressor of cell growth) upregulation. In this study, we have assessed how adipose-derived stem cells (ADSCs) affect the expansion of hematopoietic stem cells (HSCs), as well as miR-145 and p53 expressions.

Materials and methods: In this experimental study, we cultured passage-3 isolated human ADSCs as a feeder layer. Flow cytometry analysis confirmed the presence of ADSC surface markers CD73, CD90, CD105. Ex vivo cultures of cordblood CD34+ cells were cultured under the following 4 culture conditions for 7 days: i. Medium only supplemented with cytokines, ii. Culture on an ADSCs feeder layer, iii. Indirect culture on an ADSCs feeder layer (Thin Cert™ plate with a 0.4 μm pore size), and iv. Control group analyzed immediately after extraction. Real-time polymerase chain reaction (PCR) was used to determine the expressions of the p53 and miR-145 genes. Flow cytometry analysis of cells stained by annexin V and propidium iodide (PI) was performed to detect the rate of apoptosis in the expanded cells.

Results: ADSCs tested positive for mesenchymal stem cell (MSC) markers CD105, CD90, and CD73, and negative for HSC markers CD34 and CD45. Our data demonstrated the differentiation potential of ASCs to osteoblasts by alizarin red and alkaline phosphatase staining. MTT assay results showed a higher proliferation rate of CD34+cells directly cultured on the ADSCs feeder layer group compared to the other groups. Direct contact between HSCs and the feeder layer was prevented by a microporous membrane p53 expression increased in the HSCs group with indirect contact of the feeder layer compared to direct contact of the feeder layer. p53 significantly downregulated in HSCs cultured on ADSCs, whereas miR-145 significantly upregulated in HSCs cultured on ADSCs.

Conclusions: ADSCs might increase HSCs proliferation and self-renewal through miR-145, p53, and their relationship.

Keywords: Adipose Cell; Hematopoietic Stem Cell; MicroRNA.

Fig.1

Flow cytometry analysis of adipose-derived stem cell (ADSCs), markers showed positive expressions of 98.4% of ADSCs, CD105⁺, 87.3% cells are CD73⁺, 80.5% are CD90 and 0.303% are CD45 positive.

Fig.2

Cultured hematopoietic stem cells on an adipose-derived stem cell feeder layer. A. After 2 days and B. After 7 days.

Fig.3

Osteogenic differentiation of adipose-derived stem cell, 200. A. Positive reaction in osteoblastic differentiated cells with alizarin red staining, B. Undifferentiated cells, C. Osteoblast differentiated cells with increased alkaline phosphatase activity, and D. Undifferentiated cells.

Fig.4

Analysis of p53 and miR-145 expressions by reverse transcription PCR in A, B, C, and D groups. A; Fresh CD34⁺ cells, B; CD34⁺ cells cultured in the presence of cytokines, C; CD34⁺ cells indirectly cultured on feeder layer, and D; CD34⁺ cells directly cultured on feeder layer. GAPDH group was considered the control group.

Fig.5

Analysis of p53 gene expression in fresh CD34⁺ cells by real-time polymerase chain reaction compared to the other groups. A; p53 gene expression in fresh CD34⁺ cells, B; Expression of p53 in CD34⁺ cells in the presence of cytokines, C; Expression of p53 in CD34⁺ cells indirectly cultured on the feeder layer, D; Expression of p53 in CD34⁺ cells directly cultured on the feeder layer. Fresh CD34⁺ cells showed significant increase in p53 gene expression compared to the other groups, and *; P<0.05.

Fig.6

Analysis of miR-145 expression in fresh CD34⁺ cells by real-time polymerase chain reaction compared to the other groups. A; miR-145 expression in fresh CD34⁺ cells, B; Expression of miR-145 in CD34⁺ cells in the presence of cytokines, C; Expression of miR-145in CD34⁺ cells indirectly cultured on the feeder layer, D; Expression of miR-145 in CD34⁺ cells directly cultured on the feeder layer. Fresh CD34⁺ cells showed significant increase in miR-145 expression compared to the other groups, and *; P<0.05.