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

Moharram Ahmadnejad; Naser Amirizadeh; Roya Mehrasa; Ahmad Karkhah; Mahin Nikougoftar; Arezoo Oodi

doi:10.5045/br.2017.52.1.25

Quick links

Most Cited Most Read Ahead of Print Archives Go to E-submission

Original Article

Split Viewer

Blood Res 2017; 52(1):

Published online March 27, 2017

https://doi.org/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

Moharram Ahmadnejad¹, Naser Amirizadeh¹, Roya Mehrasa¹, Ahmad Karkhah^2,3, Mahin Nikougoftar¹, and Arezoo Oodi^1*

Author Info. +

¹Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.

²Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran.

³Student Research Committee, School of Medicine, Babol University of Medical Sciences, Babol, Iran.

Correspondence to : Correspondence to Arezoo Oodi, Ph.D. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Hemmat Exp. Way, Next to the Milad Tower, 14665-1157 Tehran, Iran. a.oodi@ibto.ir

Received: July 31, 2016; Revised: December 7, 2016; Accepted: December 26, 2016

Abstract

Go to

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.

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

Article

Original Article

Blood Res 2017; 52(1): 25-30

Published online March 27, 2017 https://doi.org/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

Moharram Ahmadnejad¹, Naser Amirizadeh¹, Roya Mehrasa¹, Ahmad Karkhah^2,3, Mahin Nikougoftar¹, and Arezoo Oodi^1*

¹Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.

²Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran.

³Student Research Committee, School of Medicine, Babol University of Medical Sciences, Babol, Iran.

Correspondence to:Correspondence to Arezoo Oodi, Ph.D. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Hemmat Exp. Way, Next to the Milad Tower, 14665-1157 Tehran, Iran. a.oodi@ibto.ir

Received: July 31, 2016; Revised: December 7, 2016; Accepted: December 26, 2016

Abstract

Background

Methods

Results

Conclusion

Keywords: DNA methyltransferase 1, HSCs, MSCs, Co-culture, Ex vivo expansion, Cytokines

Abstract

Fig 1.

Download

Figure 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).

Blood Research 2017; 52: 25-30https://doi.org/10.5045/br.2017.52.1.25

Fig 2.

Download

Figure 2.

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

Blood Research 2017; 52: 25-30https://doi.org/10.5045/br.2017.52.1.25

Fig 3.

Download

Figure 3.

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

Blood Research 2017; 52: 25-30https://doi.org/10.5045/br.2017.52.1.25

Fig 4.

Download

Figure 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.

Blood Research 2017; 52: 25-30https://doi.org/10.5045/br.2017.52.1.25