Blood Res 2013; 48(2):
Published online June 25, 2013
https://doi.org/10.5045/br.2013.48.2.107
© The Korean Society of Hematology
Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Correspondence to : Correspondence to Hong Hoe Koo, M.D., Ph.D. Department of Pediatrics, Samsung Medical Center, 81, Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. Tel: +82-2-3410-3524, Fax: +82-2-3410-0043, hhkoo@skku.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times.
Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay.
The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), α4-integrin, α6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-γ in these cells.
We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, α6-integrin, α4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore,
Keywords Mesenchymal stem cell, Gene expression pattern, Seeding density, Culture time, Cell therapy
Blood Res 2013; 48(2): 107-114
Published online June 25, 2013 https://doi.org/10.5045/br.2013.48.2.107
Copyright © The Korean Society of Hematology.
Myoung Woo Lee#, Dae Seong Kim#, Keon Hee Yoo*, Hye Ryung Kim, In Keun Jang, Ji Hyang Lee, So Yeon Kim, Meong Hi Son, Soo Hyun Lee, Hye Lim Jung, Ki Woong Sung, and Hong Hoe Koo*
Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Correspondence to:Correspondence to Hong Hoe Koo, M.D., Ph.D. Department of Pediatrics, Samsung Medical Center, 81, Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. Tel: +82-2-3410-3524, Fax: +82-2-3410-0043, hhkoo@skku.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times.
Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay.
The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), α4-integrin, α6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-γ in these cells.
We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, α6-integrin, α4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore,
Keywords: Mesenchymal stem cell, Gene expression pattern, Seeding density, Culture time, Cell therapy
Characteristics of MSCs isolated from human bone marrow.
Phase-contrast micrograph of BM-MSCs obtained from 5-, 10-, and 15-day cultures after seeding at a density of 200, 1,000, and 5,000 cells/cm2 (×40). BM-MSCs seeded at a density of 200, 1,000, and 5,000 cells/cm2 were observed after 5, 10, and 15 days in culture with an inverted microscope.
Cell density of MSCs obtained from 5-, 10-, and 15-day cultures after seeding at a density of 200, 1,000, and 5,000 cells/cm2. Doubling numbers
RT-PCR analysis of MSCs after seeding at different densities and culturing for varying times. Total RNA of MSCs, obtained at 5, 10, and 15 days after plating at 200, 1,000, and 5,000 cells/cm2, was analyzed by RT-PCR with primers specific for PODXL, α6-integrin, α4-integrin, LIF, CXCR4, and HGF. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to
Stemness gene expression by MSCs after seeding at 200 cells/cm2. Total RNA of MSCs, obtained at 5-, 10-, and 15-day cultures after plating at a density of 200 cells/cm2, was analyzed by RT-PCR with primers specific for Oct-4 and Nanog. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to
Expression levels of immunomodulatory factors in MSCs cultured for 10 days after seeding at 200 cells/cm2. MSCs were seeded at a density of 200 cells/cm2 and cultured for 10 days, and then treated with or without IFN-γ for 48 h. Total RNA of these MSCs was analyzed by RT-PCR with primers specific for IDO, IL-10, HGF, COX-1, and COX-2. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to
Table 1 . Primer sequences for RT-PCR analysis..
a)Forward (F) and reverse (R) primers used to detect mRNA expression of the indicated targets..
Abbreviations: PODXL, Podocalyxin-like protein; LIF, leukemia inhibitory factor; CXCR4, C-X-C chemokine receptor type 4; IDO, indoleamine 2,3-dioxygenase; IL-10, interleukin-10; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; HGF, hepatocyte growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase..
Woan-Sang Kim, Sangho Lee, and Young-sup Yoon
Blood Res 2013; 48(2): 76-86Ji Young Lim, Bo Gyeong Kim, Seol Kyung Moon, Chang Ki Min
Korean J Hematol 2008; 43(4): 219-231Sang rhim Choi, Dae Hyeoung Lee, Dae Chul Jeong, Hui Sung Hwang, Nack Gyun Chung, Bin Cho, Chi Wha Han, Hack Ki Kim.
Korean J Hematol 2006; 41(4): 250-258
Characteristics of MSCs isolated from human bone marrow.
Phase-contrast micrograph of BM-MSCs obtained from 5-, 10-, and 15-day cultures after seeding at a density of 200, 1,000, and 5,000 cells/cm2 (×40). BM-MSCs seeded at a density of 200, 1,000, and 5,000 cells/cm2 were observed after 5, 10, and 15 days in culture with an inverted microscope.
|@|~(^,^)~|@|Cell density of MSCs obtained from 5-, 10-, and 15-day cultures after seeding at a density of 200, 1,000, and 5,000 cells/cm2. Doubling numbers
RT-PCR analysis of MSCs after seeding at different densities and culturing for varying times. Total RNA of MSCs, obtained at 5, 10, and 15 days after plating at 200, 1,000, and 5,000 cells/cm2, was analyzed by RT-PCR with primers specific for PODXL, α6-integrin, α4-integrin, LIF, CXCR4, and HGF. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to
Stemness gene expression by MSCs after seeding at 200 cells/cm2. Total RNA of MSCs, obtained at 5-, 10-, and 15-day cultures after plating at a density of 200 cells/cm2, was analyzed by RT-PCR with primers specific for Oct-4 and Nanog. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to
Expression levels of immunomodulatory factors in MSCs cultured for 10 days after seeding at 200 cells/cm2. MSCs were seeded at a density of 200 cells/cm2 and cultured for 10 days, and then treated with or without IFN-γ for 48 h. Total RNA of these MSCs was analyzed by RT-PCR with primers specific for IDO, IL-10, HGF, COX-1, and COX-2. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to