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
Mesenchymal stem cells (MSCs) are known to have significant potential for cell therapies, including cell-based transplantations and regenerative medicine, owing to their homing ability, engraftment efficiency, and therapeutic value following a homing event, as evidenced by differentiation into tissue-specific cell types [1, 2] and potent immunosuppressive effects [3, 4]. However, the results have been variable in part, which may reflect the intrinsic heterogeneity of MSCs, as well as differences in culture conditions for isolated MSCs.
MSCs exhibit heterogeneous characteristics with regard to morphology, proliferation rate, and secreted factors. Previous studies have attempted to characterize the heterogeneity of these cells, and preclinical trials using cells based on the intrinsic heterogeneity have been conducted [5-9]. Numerous attempts have been made to develop more specific procedures for the isolation and preparation of appropriate cells from this heterogeneous cell population. In particular, ex vivo expansion of MSCs is used for developing and maintaining the cells used for cell therapy. The methods used to expand and characterize the cells are critical factors in preparing MSCs. However, adequate protocols for preparing and characterizing MSCs have not yet been standardized. Although
Podocalyxin-like protein (PODXL), α6-integrin, and α4-integrin are known to contribute to hematopoietic stem cell homing to the bone marrow (BM) [13], as well as the clonogenicity of MSCs and their migration to damaged tissues [5]. Leukemia inhibitory factor (LIF) also regulates humoral and cellular immune responses in damaged tissues [14] and induces the
In this study, we explored differences in the expression of PODXL, α4-integrin, α6-integrin, and LIF genes that play a crucial role in cell migration and tissue regeneration in BM-MSCs expanded under various seeding densities and cultivation times. In addition, we confirmed the expression of stemness and immunomodulatory genes in BM-MSCs under appropriate culture conditions under which those genes were highly expressed.
The Institutional Review Board of Samsung Medical Center approved this study. After obtaining written informed consent from the patients, we obtained iliac crest BM aspirates from normal adult volunteers. Mononuclear cells were isolated from normal BM aspirates by using Ficoll-Hypaque (Histopaque-1077; Sigma-Aldrich, St. Louis, MO) density gradient centrifugation. Cells were plated at 3×105 cells/cm2 in low glucose Dulbecco's Modified Eagle's Medium (LG-DMEM; Invitrogen-Gibco, Rockville, MD) containing 10% fetal bovine serum (FBS; Invitrogen-Gibco) and 100 U/mL penicillin/streptomycin (Invitrogen-Gibco). After 24 h, non-adherent cells were removed. Adherent cells were cultured for an additional 5-10 days, until they were ~70% confluent.
Antibodies against the human antigens CD14, CD34, CD45, CD73, and CD90 were purchased from Becton Dickinson (Franklin Lakes, NJ). Antibodies against CD105 were purchased from Ancell (Bayport, MN). A total of 5×105 cells were resuspended in 0.2 mL PBS and incubated with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies for 20 min at room temperature. The fluorescence intensity of the cells was evaluated by flow cytometry (FACScan; Becton Dickinson), and data were analyzed with CELLQUEST software (Becton Dickinson).
First passage MSCs were plated at 5×105 cells/well in 6-well plates in LG-DMEM containing 10% FBS, allowed to adhere overnight, and replaced with LG-DMEM containing 10% FBS supplemented with 0.1 µM dexamethasone (Sigma-Aldrich), 10 mM β-glycerolphosphate (Sigma-Aldrich), and 100 µM ascorbate-2-phosphate (Sigma-Aldrich). The medium was changed every 3 days. After 14-21 days, osteoblast differentiation was determined by alkaline phosphatase expression.
As described above, cells were cultured for 14-21 days after reaching confluence in LG-DMEM containing 10% FBS, 1 µM dexamethasone, 500 µM isobutyl methylxanthine (Sigma-Aldrich), 100 µM indomethacin (Sigma-Aldrich), and 10 µg/mL insulin (Sigma-Aldrich). Adipogenic differentiation was evaluated by detecting cellular accumulation of neutral lipid vacuoles via staining with Oil-red O (Sigma-Aldrich) solution.
A total of 1×106 cells were pelleted in a 15-mL tube by centrifugation at 300 ×
Second-passage MSCs were used throughout these studies. MSCs seeded at a density of 200, 1,000, and 5,000 cells/cm2 were observed on day 5, 10, and 15 of culture with an inverted microscope (Olympus CK40; Olympus, Melville, NY). The total cell number of MSCs was determined using a hematocytometer (Marienfeld, Germany).
Viable first-passage MSCs were plated at 200, 1,000, and 5,000 cells/cm2 and incubated for 5, 10, and 15 days each. To obtain interferon (IFN)-γ-treated MSCs, MSCs plated at 200 cells/cm2 that were cultured for 10 days were incubated in the presence of 200 IU/mL IFN-γ (R&D Systems) for 48 h. Total RNA was isolated from 106 cells by using the QIAGEN RNeasy Mini Kit (Qiagen, Valencia, CA), which was used to perform semi-quantitative reverse-transcription PCR assays with a commercial kit (PrimeScript 1st strand cDNA synthesis kit; TaKaRa Shuzo, Shiga, Japan). Band intensity was measured using NIH image/ImageJ and normalized against that of
Data are expressed as the mean±standard deviation.
Human BM-derived MSCs were fibroblastic in shape. The cell surface antigen profiles of these cells after 2 passages in culture were analyzed by flow cytometry. The cells were positive for MSC-specific surface markers such as CD73, CD90, and CD105, but negative for CD14 and CD45 (Fig. 1A). The cells also exhibited mesenchymal differentiation potential, as assessed by culturing cells in osteogenic, chondrogenic, or adipogenic medium (Fig. 1B). Viable first-passage MSCs were plated at 200, 1,000, and 5,000 cells/cm2 and incubated for 5, 10, and 15 days (Fig. 2), and their yields were determined (Fig. 3). Cultures plated at a density of 200 cells/cm2 were ~50% confluent (7,800±1,200 cells/cm2) after 10 days, and most cells were thin and spindle-shaped. Cultures plated at 1,000 cells/cm2 were ~100% confluent (approximately 9,500±1,400 to 42,000±3,100 cells/cm2) after 5-15 days of culture. Cultures plated at 5,000 cells/cm2 were over-confluent, such that the cell yields were approximately 24,300±4,700 to 48,400±4,900 cells/cm2 after 515 days, and most cells showed extensive cell-to-cell contacts.
PODXL, α4-integrin, α6-integrin, and LIF are all linked to cell motility and migration [13, 17, 18]. After verifying the mRNA expression of PODXL, α4-integrin, α6-integrin, and C-X-C chemokine receptor type 4 (CXCR4) in MSCs plated at 500 cells/cm2, which is equivalent to a 50% confluent seeding density, the gene expression levels of MSCs plated at low or high densities were examined. RT-PCR revealed that PODXL, α4-integrin, α6-integrin, and LIF transcripts were present at the highest levels in 10-day MSC cultures seeded at a density of 200 cells/cm2 (Fig. 4). The PODXL mRNA levels were the highest in 10-day MSC cultures seeded at densities of 200 or 1,000 cells/cm2. The mRNA levels of α4-integrin, α6-integrin, and LIF were highest in cultures seeded at 200 cells/cm2, which were reduced when the cells were seeded at 1,000 or 5,000 cells/cm2. The mRNA levels of CXCR4 and hepatocyte growth factor (HGF) were not significantly different under any culture conditions.
RT-PCR assays showed that
The heterogeneity of MSCs and differences in conditions used to culture MSCs may be the main causes of the contradictory results from numerous studies and clinical trials. As a result, the methods by which the cells are expanded in culture and characterized are critical factors in terms of preparing MSCs with the desired features. By expanding MSCs
MSCs expanded
Damaged tissues require the stemness and potent immunomodulatory properties of MSCs as a component of the therapeutic action of MSCs following a homing event for tissue reconstitution. The observed expression of Oct-4 and Nanog in 10-day cultures after seeding at a density of 200 cells/cm2 shows stem cell maintenance in the adult organism that is essential for tissue homeostasis and for initiation of native tissue regeneration and responses to injury [22]. Therefore, the identification of regulatory factors that maintain the "stemness" properties of MSCs is important for the practical purpose of producing sufficient quantities of MSCs for therapeutic applications. The anti-inflammation and immunomodulation exhibited by MSCs in their initiation of native tissue regeneration may also be important for tissue reconstitution in therapeutic settings. The inhibitory effect of MSCs on human mononuclear cell proliferation [20] and immunomodulatory gene expression of cultured MSCs when seeded at a low density indicates that MSCs retain their immunomodulatory properties in an inflammatory environment (i.e., in the presence of IFN-γ); this increases the possibility of protecting target stem cells and tissues, thereby supporting a possible use for MSCs in regenerative therapy under inflammatory conditions.
In conclusion, our results showed differences in the gene expression patterns of MSCs according to the seeding density and culture time. MSCs from 10-day cultures after seeding at a low density were efficiently expanded, expressed PODXL, α6-integrin, α4-integrin, and LIF, and retained their stemness and immunomodulatory gene expression. These data indicate that the seeding density and culture time are key factors in preparing MSCs for use in regenerative medicine. This study provides promising information on culture conditions appropriate for obtaining MSCs for 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.
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
Mesenchymal stem cells (MSCs) are known to have significant potential for cell therapies, including cell-based transplantations and regenerative medicine, owing to their homing ability, engraftment efficiency, and therapeutic value following a homing event, as evidenced by differentiation into tissue-specific cell types [1, 2] and potent immunosuppressive effects [3, 4]. However, the results have been variable in part, which may reflect the intrinsic heterogeneity of MSCs, as well as differences in culture conditions for isolated MSCs.
MSCs exhibit heterogeneous characteristics with regard to morphology, proliferation rate, and secreted factors. Previous studies have attempted to characterize the heterogeneity of these cells, and preclinical trials using cells based on the intrinsic heterogeneity have been conducted [5-9]. Numerous attempts have been made to develop more specific procedures for the isolation and preparation of appropriate cells from this heterogeneous cell population. In particular, ex vivo expansion of MSCs is used for developing and maintaining the cells used for cell therapy. The methods used to expand and characterize the cells are critical factors in preparing MSCs. However, adequate protocols for preparing and characterizing MSCs have not yet been standardized. Although
Podocalyxin-like protein (PODXL), α6-integrin, and α4-integrin are known to contribute to hematopoietic stem cell homing to the bone marrow (BM) [13], as well as the clonogenicity of MSCs and their migration to damaged tissues [5]. Leukemia inhibitory factor (LIF) also regulates humoral and cellular immune responses in damaged tissues [14] and induces the
In this study, we explored differences in the expression of PODXL, α4-integrin, α6-integrin, and LIF genes that play a crucial role in cell migration and tissue regeneration in BM-MSCs expanded under various seeding densities and cultivation times. In addition, we confirmed the expression of stemness and immunomodulatory genes in BM-MSCs under appropriate culture conditions under which those genes were highly expressed.
The Institutional Review Board of Samsung Medical Center approved this study. After obtaining written informed consent from the patients, we obtained iliac crest BM aspirates from normal adult volunteers. Mononuclear cells were isolated from normal BM aspirates by using Ficoll-Hypaque (Histopaque-1077; Sigma-Aldrich, St. Louis, MO) density gradient centrifugation. Cells were plated at 3×105 cells/cm2 in low glucose Dulbecco's Modified Eagle's Medium (LG-DMEM; Invitrogen-Gibco, Rockville, MD) containing 10% fetal bovine serum (FBS; Invitrogen-Gibco) and 100 U/mL penicillin/streptomycin (Invitrogen-Gibco). After 24 h, non-adherent cells were removed. Adherent cells were cultured for an additional 5-10 days, until they were ~70% confluent.
Antibodies against the human antigens CD14, CD34, CD45, CD73, and CD90 were purchased from Becton Dickinson (Franklin Lakes, NJ). Antibodies against CD105 were purchased from Ancell (Bayport, MN). A total of 5×105 cells were resuspended in 0.2 mL PBS and incubated with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies for 20 min at room temperature. The fluorescence intensity of the cells was evaluated by flow cytometry (FACScan; Becton Dickinson), and data were analyzed with CELLQUEST software (Becton Dickinson).
First passage MSCs were plated at 5×105 cells/well in 6-well plates in LG-DMEM containing 10% FBS, allowed to adhere overnight, and replaced with LG-DMEM containing 10% FBS supplemented with 0.1 µM dexamethasone (Sigma-Aldrich), 10 mM β-glycerolphosphate (Sigma-Aldrich), and 100 µM ascorbate-2-phosphate (Sigma-Aldrich). The medium was changed every 3 days. After 14-21 days, osteoblast differentiation was determined by alkaline phosphatase expression.
As described above, cells were cultured for 14-21 days after reaching confluence in LG-DMEM containing 10% FBS, 1 µM dexamethasone, 500 µM isobutyl methylxanthine (Sigma-Aldrich), 100 µM indomethacin (Sigma-Aldrich), and 10 µg/mL insulin (Sigma-Aldrich). Adipogenic differentiation was evaluated by detecting cellular accumulation of neutral lipid vacuoles via staining with Oil-red O (Sigma-Aldrich) solution.
A total of 1×106 cells were pelleted in a 15-mL tube by centrifugation at 300 ×
Second-passage MSCs were used throughout these studies. MSCs seeded at a density of 200, 1,000, and 5,000 cells/cm2 were observed on day 5, 10, and 15 of culture with an inverted microscope (Olympus CK40; Olympus, Melville, NY). The total cell number of MSCs was determined using a hematocytometer (Marienfeld, Germany).
Viable first-passage MSCs were plated at 200, 1,000, and 5,000 cells/cm2 and incubated for 5, 10, and 15 days each. To obtain interferon (IFN)-γ-treated MSCs, MSCs plated at 200 cells/cm2 that were cultured for 10 days were incubated in the presence of 200 IU/mL IFN-γ (R&D Systems) for 48 h. Total RNA was isolated from 106 cells by using the QIAGEN RNeasy Mini Kit (Qiagen, Valencia, CA), which was used to perform semi-quantitative reverse-transcription PCR assays with a commercial kit (PrimeScript 1st strand cDNA synthesis kit; TaKaRa Shuzo, Shiga, Japan). Band intensity was measured using NIH image/ImageJ and normalized against that of
Data are expressed as the mean±standard deviation.
Human BM-derived MSCs were fibroblastic in shape. The cell surface antigen profiles of these cells after 2 passages in culture were analyzed by flow cytometry. The cells were positive for MSC-specific surface markers such as CD73, CD90, and CD105, but negative for CD14 and CD45 (Fig. 1A). The cells also exhibited mesenchymal differentiation potential, as assessed by culturing cells in osteogenic, chondrogenic, or adipogenic medium (Fig. 1B). Viable first-passage MSCs were plated at 200, 1,000, and 5,000 cells/cm2 and incubated for 5, 10, and 15 days (Fig. 2), and their yields were determined (Fig. 3). Cultures plated at a density of 200 cells/cm2 were ~50% confluent (7,800±1,200 cells/cm2) after 10 days, and most cells were thin and spindle-shaped. Cultures plated at 1,000 cells/cm2 were ~100% confluent (approximately 9,500±1,400 to 42,000±3,100 cells/cm2) after 5-15 days of culture. Cultures plated at 5,000 cells/cm2 were over-confluent, such that the cell yields were approximately 24,300±4,700 to 48,400±4,900 cells/cm2 after 515 days, and most cells showed extensive cell-to-cell contacts.
PODXL, α4-integrin, α6-integrin, and LIF are all linked to cell motility and migration [13, 17, 18]. After verifying the mRNA expression of PODXL, α4-integrin, α6-integrin, and C-X-C chemokine receptor type 4 (CXCR4) in MSCs plated at 500 cells/cm2, which is equivalent to a 50% confluent seeding density, the gene expression levels of MSCs plated at low or high densities were examined. RT-PCR revealed that PODXL, α4-integrin, α6-integrin, and LIF transcripts were present at the highest levels in 10-day MSC cultures seeded at a density of 200 cells/cm2 (Fig. 4). The PODXL mRNA levels were the highest in 10-day MSC cultures seeded at densities of 200 or 1,000 cells/cm2. The mRNA levels of α4-integrin, α6-integrin, and LIF were highest in cultures seeded at 200 cells/cm2, which were reduced when the cells were seeded at 1,000 or 5,000 cells/cm2. The mRNA levels of CXCR4 and hepatocyte growth factor (HGF) were not significantly different under any culture conditions.
RT-PCR assays showed that
The heterogeneity of MSCs and differences in conditions used to culture MSCs may be the main causes of the contradictory results from numerous studies and clinical trials. As a result, the methods by which the cells are expanded in culture and characterized are critical factors in terms of preparing MSCs with the desired features. By expanding MSCs
MSCs expanded
Damaged tissues require the stemness and potent immunomodulatory properties of MSCs as a component of the therapeutic action of MSCs following a homing event for tissue reconstitution. The observed expression of Oct-4 and Nanog in 10-day cultures after seeding at a density of 200 cells/cm2 shows stem cell maintenance in the adult organism that is essential for tissue homeostasis and for initiation of native tissue regeneration and responses to injury [22]. Therefore, the identification of regulatory factors that maintain the "stemness" properties of MSCs is important for the practical purpose of producing sufficient quantities of MSCs for therapeutic applications. The anti-inflammation and immunomodulation exhibited by MSCs in their initiation of native tissue regeneration may also be important for tissue reconstitution in therapeutic settings. The inhibitory effect of MSCs on human mononuclear cell proliferation [20] and immunomodulatory gene expression of cultured MSCs when seeded at a low density indicates that MSCs retain their immunomodulatory properties in an inflammatory environment (i.e., in the presence of IFN-γ); this increases the possibility of protecting target stem cells and tissues, thereby supporting a possible use for MSCs in regenerative therapy under inflammatory conditions.
In conclusion, our results showed differences in the gene expression patterns of MSCs according to the seeding density and culture time. MSCs from 10-day cultures after seeding at a low density were efficiently expanded, expressed PODXL, α6-integrin, α4-integrin, and LIF, and retained their stemness and immunomodulatory gene expression. These data indicate that the seeding density and culture time are key factors in preparing MSCs for use in regenerative medicine. This study provides promising information on culture conditions appropriate for obtaining MSCs for 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