Expression of hyaluronan synthase genes in umbilical cord blood stem/progenitor cells

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Abstract

Scientific progress reveals an ever-expanding role of hyaluronan (HA) in diverse biological functions. It has become increasingly clear that HA might also be essential for certain functions of stem cells. CD133+ cells isolated from umbilical cord blood (UCB) seem to represent an alternative to CD34+ cells as a source of transplantable haematopoietic progenitor cells. The aim of this study was to investigate expression patterns of hyaluronan synthases (HAS) genes in freshly isolated and cultured UCB progenitor cells and to compare HAS mRNA levels to those found in non-progenitor cells. CD133+ stem cells were isolated from UCB using an immunomagnetic procedure. Investigation of HAS mRNA expression patterns in CD133+ and CD133− cells by RT-PCR was performed immediately after isolation as well as after cultivation towards myelomonocytic lineage. In addition, activation patterns of mitogen activated protein kinases (MAPK) were analyzed by Western blot experiments. mRNA for HAS1 is undetectable but HAS3 mRNA can be readily detected in freshly isolated CD133+ as well as in CD133− UCB cells. More importantly, our data demonstrate that mRNA for HAS2 can only be detected in CD133+ progenitor cells. In addition, while MAPK are slightly activated in CD133− UCB cells, no significant phosphorylation of MAPK could be observed in CD133+ cells, excluding a role of these kinases in the regulation of HAS2. HAS2 is expressed only in freshly isolated CD133+ cells and quickly diminishes during differentiation. Because of this, HAS2 gene expression might be suitable as a new marker for CD133+ UCB-derived stem cells.

Introduction

Hyaluronan (HA), a major component of the extracellular matrix, plays a role in cell proliferation, cell migration, inflammation, tumorigenesis, angiogenesis and also in the embryonic development [1], [2]. Three hyaluronan synthase (HAS) genes encoding for a plasma membrane protein are responsible for HA synthesis. HAS isoforms not only differ in their enzymatic properties, but also in their kinetic characteristics and in product size. In mammalian cells, the expression of the HAS isoforms in response to various cytokines is controlled by diverse mechanisms [3], [4]. Furthermore, there are clearly differences in the expression patterns of these genes, which are differently regulated, both spatially and temporally, during embryonic development. Knockout experiments have shown that HAS2(−/−) embryos lack hyaluronan (HA); such animals exhibit severe cardiac and vascular abnormalities and die during mid gestation [5]. Heart explants from HAS2(−/−) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. Interestingly, double HAS1/HAS3 knockout embryos are viable and without phenotypic changes [5].

Whether and to what degree these genes also play a dominant role in the biology of stem cells is less clear. Nevertheless, the importance of the HAS genes in embryogenesis led us to hypothesize that HAS2 may be of importance in stem cell biology as well. Human umbilical cord blood is a relevant source of progenitor cells (PC). Here, we describe experiments investigating the expression pattern of the HAS genes in cells isolated from umbilical cord blood. HAS expression patterns were monitored in freshly isolated PC and compared to mRNA levels of these genes in non-progenitor cells (non-PC). In addition, changes in mRNA levels of these genes were monitored for several weeks.

With the exception of chemokine/cytokine-induced HAS1 activation in synoviocytes, there are no reports published, investigating intracellular signaling pathways leading to the activation of HAS genes. We were the first to demonstrate the requirement for certain MAPK in TGF-β induced HAS activation in synoviocytes [3], [6]. We also found that in synoviocytes, the activation of the transcription factor NF-κB is essential for IL-1β induced HAS1 mRNA transcription [7]. In an attempt to gain insight into the intracellular signaling mechanisms involved in the regulation of these genes in stem cells, we also performed western blot experiments investigating the activation of mitogen activated protein kinases (MAPK) (stress activated protein kinase (SAPK)/c-Jun amino-terminal kinase (JNK), extracellular signal activated protein kinase (ERK), and p38 protein kinase) in PC and compared results with data obtained on non-PC.

Section snippets

Isolation of cells

Human umbilical cord blood (UCB) cells were obtained from full-term normal deliveries with informed consent. The mononuclear cells (MNC) fraction was separated by density centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Freiburg, Germany). CD133+ (PC) cells were isolated using CD133-conjugated super paramagnetic micro beads and MiniMACS columns (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) according to manufacturer's instructions. Briefly, MNC were incubated with CD133

Total cell expansion of PC (CD133 + CD117 + CD34 + Lin− stem cells) and differentiation towards myelomonocytic lineage

We demonstrated in a recent article that under certain conditions CD133 + CD117 + CD34 + Lin− UCB stem/progenitor cells differentiate towards myelomonocytic lineage [10]. We further documented that these stem cells expressed the highest level of CD14 and CD15 surface markers when cultivated in media containing 12.5% horse serum, 12.5% FBS, 100 ng/ml stem cell growth factor and 50 ng/ml IL-3 [10]. UCB cells in the experiments described in this manuscript were isolated and differentiated as described

Discussion

Stem cell technology seems to offer unprecedented opportunities for the treatment of many ailments. As a consequence, enormous efforts have been undertaken to define genes that are essential for stem cell function as well as in finding markers for stem cells that will facilitate isolation and characterization of stem cell populations. Highly sophisticated methods such as a gene knockout models and screening tools like genome wide micro-arrays are increasingly used to identify genes that are

Conclusions

Taken together, extensive research is still needed for a better understanding of the relevance of HAS2 in general—but even more so for a better understanding of the unique biological function of HAS2 in UCB derived PC. Nevertheless, the demonstration that in CD133-positive UCB cells HAS2 is expressed only in freshly isolated PC might serve as a novel marker for these cell type. In addition, this phenomenon might also be suited to conceive novel ways for isolation and enrichment of this cell

Acknowledgements

This work was supported in part by grants from the City of Vienna, the Austrian Ministry of Health and Women (236065/008-I/A/Ib/2004), the Austrian Ministry of Education, Science and Culture, and the Austrian National Bank (Jubilaeumsfonds No. 10847).

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