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

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.

Rapid Isolation of Adipose Tissue-Derived Stem Cells by the Storage of Lipoaspirates

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.

Difference in Viability of CD34+Cells in Cryopreserved Cord Blood According to Evaluation Methods / 대한혈액학회지

BACKGROUND: On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, accurate evaluation of viability of CD34+cells in cryopreserved UCB has clinical implication. We examined the difference in viability of cells in cryopreserved UCB according to the duration of cryopreservation and different methods. METHODS: A total of 60 UCB samples which were cryopreserved for 1 to 4 years were used in this study. Viability of cryopreserved cells were examined with trypan blue exclusion assay, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining. RESULTS: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+cells were viable as identified by trypan blue exclusion assay. In the CD34+cell population, the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis. However, cspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10~20% at 2, 4, and 6 hours after thawing. CONCLUSION: Viable cells in UCB should be measured by several compensatory techniques rather than a single method. Discordance among Annexin-V/PI staining versus trypan blue exclusion, DNA contents analysis, and the caspase-3 activation test or intracellular esterase activity should be clarified in order to apply these techniques for actual cord blood transplantation.

Effect of the Antigenic Differences of Neuroblastoma on the Maturation and Immunoactivity in Dendritic Cells / 대한소아혈액종양학회지

PURPOSE: Most of neuroblastoma (NB) patients present with advanced disease, and the survival of patients with advanced stage disease remains poor, despite of aggressive therapy such as high dose chemotherapy and autologouse stem cell transplantation. It is necessary to control of minimal residual disease in NB patients in order to reduce relapse rate. Dendritic cells (DC) are crucial for induction of antitumor immunity. Recent studies suggest that tumors avoid immune surveillence by inhibiting DC function. We investigated the effect of NB cells about maturation and/or function of dendritic cells using peripheral blood monocytes as dendritic cell source. METHODS: DCs were generated in the presence of GM-CSF (granulocyte and marcrophage colony-stimulating factor) and IL (interleukin) -4 from peripheral blood of healthy donors. DCs were exposed to human some kinds of NB cells or NB lysate. And the maturation of DC was induced by adding TNF (tumor necrosis factor) -alpha, IL-1beta, IL-6 and prostaglandin E2 for 2 days. DCs were analyzed by flow cytometry and mixed lymphocyte reactions. RESULTS: DCs exposed to NB cells didn't upregulate the expression of CD83, HLA-DR, CD80 and CD86, and DCs exposed to NB lysate didn't upregulate the expression of CD83 and HLA-DR. DCs exposed to NB cells and NB lysate inhibited the proliferation of allogenic T cells in mixed lymphocyte reactions. CONCLUSION: NB cells induced impaired maturation and immune function of DCs. These findings have significant implications for DC-based immunotherapy in the treatment NB and suggested that it was necessary to develop a new method of priming antigen to dendritic cells at NB immunotherapy.