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Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

ABSTRACT: We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3' end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from "stem cell genes" being highly expressed at 0 h and turned off at 48 h, while "cell division" genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

SUBMITTER: Lambert JF

PROVIDER: S-EPMC2193900 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

Lambert Jean-Francois JF Liu Meng M Colvin Gerald A GA Dooner Mark M McAuliffe Christina I CI Becker Pamela S PS Forget Bernard G BG Weissman Sherman M SM Quesenberry Peter J PJ

The Journal of experimental medicine 20030601 11

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of ...[more]

PMID: 12782718

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Project description:BackgroundCo-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified.MethodsMice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration.ResultsMice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro.ConclusionsCollectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

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Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

Publications

Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

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