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A subset of mobilized human hematopoietic stem cells express germ layer lineage genes which can be modulated by culture conditions.

ABSTRACT: BACKGROUND:Adult bone marrow contains stem cells that replenish the myeloid and lymphoid lineages. A subset of human and mouse CD34+ bone marrow stem cells can be propagated in culture to autonomously express embryonic stem cell genes and embryonic germ layer lineage genes. The current study was undertaken to determine whether these CD34+ stem cells could be obtained from human blood, whether gene expression could be modulated by culture conditions and whether the cells produce insulin. METHODS:Human peripheral blood buffy coat cells and mobilized CD34+ cells from human blood and from blood from C57Bl/6 J mice were cultured in hybridoma medium or neural stem cell induction medium supplemented with interleukin (IL)-3, IL-6, and stem cell factor (SCF). Changes in mRNA and protein expression were assessed by Western blot analysis and by immunohistochemistry. Mass spectrometry was used to assess insulin production. RESULTS:We were able to culture CD34+ cells expressing embryonic stem cell and embryonic germ layer lineage genes from adult human peripheral blood after standard mobilization procedures and from mouse peripheral blood. Gene expression could be modulated by culture conditions, and the cells produced insulin in culture. CONCLUSION:These results suggest a practical method for obtaining large numbers of CD34+ cells from humans to allow studies on their potential to differentiate into other cell types.

SUBMITTER: Makar T

PROVIDER: S-EPMC5930965 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.

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A subset of mobilized human hematopoietic stem cells express germ layer lineage genes which can be modulated by culture conditions.

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