Blood Res.  2017 Mar;52(1):25-30. 10.5045/br.2017.52.1.25.

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs

• Ahmadnejad M
• Amirizadeh N
• Mehrasa R
• Karkhah A
• Nikougoftar M
• Oodi A

• KMID: 2375200
• DOI: http://doi.org/10.5045/br.2017.52.1.25

Abstract

BACKGROUND
Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs.
METHODS
Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction.
RESULTS
Maximum CB-CD34⁺ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34⁺ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34⁺ and CD34⁻ cells in the cytokine co-culture system was observed.
CONCLUSION
These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

Keyword

DNA methyltransferase 1; HSCs; MSCs; Co-culture; Ex vivo expansion; Cytokines

MeSH Terms

Apoptosis
Cell Count
Coculture Techniques
Cytokines
DNA
Fetal Blood
Flow Cytometry
Hematopoietic Stem Cells*
Humans*
In Vitro Techniques
Mesenchymal Stromal Cells
Real-Time Polymerase Chain Reaction
RNA, Messenger
Stem Cells
Trypan Blue
Cytokines
DNA
RNA, Messenger
Trypan Blue

Figure

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  Fig. 1 (A) MSCs isolated from the bone marrow (×20). (B) Characterization of MSCs using flow cytometry. Evaluation of osteogenic differentiation of MSCs cultured under control conditions (C) and osteogenic conditions (D).

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  Fig. 2 The fold increase in CD34+ cells during 7 days of expansion in three culture conditions. a)This shows significant changes.

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  Fig. 3 CFU assay of expanded CD34+ cells at day 7 of culture. a)This shows significant changes.

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  Fig. 4 The fold change ratio of DNMT1 mRNA expression level in the expanded cells and in the fresh CD34+ cells in all three culture conditions (P<0.05). a)This shows significant changes.

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