Blood Res.  2013 Jun;48(2):107-114. 10.5045/br.2013.48.2.107.

Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions

• Lee MW
• Kim DS
• Yoo KH
• Kim HR
• Jang IK
• Lee JH
• Kim SY
• Son MH
• Lee SH
• Jung HL
• Sung KW
• Koo HH

• KMID: 2172908
• DOI: http://doi.org/10.5045/br.2013.48.2.107

Abstract

BACKGROUND
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.
METHODS
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.
RESULTS
The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), alpha4-integrin, alpha6-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-gamma in these cells.
CONCLUSION
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, alpha6-integrin, alpha4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.

Keyword

Mesenchymal stem cell; Gene expression pattern; Seeding density; Culture time; Cell therapy

MeSH Terms

Cell Count
Cell Movement
Cyclooxygenase 1
Gene Expression
Hepatocyte Growth Factor
Humans
Immunomodulation
Indoleamine-Pyrrole 2,3,-Dioxygenase
Interferon-gamma
Leukemia Inhibitory Factor
Mesenchymal Stromal Cells
Polymerase Chain Reaction
Population Characteristics
Regeneration
RNA, Messenger
Seeds
Tissue Therapy
Cyclooxygenase 1
Hepatocyte Growth Factor
Indoleamine-Pyrrole 2,3,-Dioxygenase
Interferon-gamma
Leukemia Inhibitory Factor
RNA, Messenger

Figure

• 

  Fig. 1 Characteristics of MSCs isolated from human bone marrow. (A) Flow cytometric analysis of MSC cell surface markers. The isolated cells were positive for CD73, CD90, and CD105 but negative for CD14, CD34, and CD45. (B) Differentiation of BM-MSCs. First passage BM-MSCs were incubated for 14-21 days in the presence of specific differentiation agents for osteoblasts, chondrocytes, and adipocytes. Alkaline phosphatase staining shows mineralization of the extracellular matrix. Toluidine blue staining shows the deposition of proteoglycans and lacunae. Differentiation into the adipocyte lineage was demonstrated by staining with Oil Red O (×200).

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  Fig. 2 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.

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  Fig. 3 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 (A) and total cell numbers (B) of MSCs obtained from 5-, 10-, and 15-day cultures after seeding at a density of 200, 1,000, and 1,000 cells/cm2. Approximately 105 MSCs were thawed, plated in 174-cm2 dishes, incubated for 4 days, and then replated at 200, 1,000, and 5,000 cells/cm2 on the following day and cultured for 5, 10, and 15 days. The number of MSCs were counted using a hematocytometer.

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  Fig. 4 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 GAPDH mRNA.

• 

  Fig. 5 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 GAPDH mRNA.

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  Fig. 6 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 GAPDH mRNA.

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