ALBERT

Description: In mucopolysaccharidosis-I (MPS-I), α-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2–FGFR1-HS interactions, resulting in defective FGF-2–induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS–cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

Description: We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1α (MIP-1α), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1α binding ability. A specific MIP-1α-binding HS oligosaccharide preparation of Mr 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1α binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.

Description: Factors responsible for long-term survival and proliferation of human hematopoietic stem cells (hHSC), and their mechanisms of action, remain to be defined. We previously showed that specific O-sulfated heparan sulfate (OS-HS) improves human long-term culture initiating cell (LTC-IC) maintenance for up to 5 wks in vitro. This is related to the ability of OS-HS to bind to and modulate the activity of heparin-binding cytokines on primitive hematopoietic progenitors (PHP). Recent studies indicate that bone morphogenetic proteins (BMPs) influence the development of embryonic hematopoiesis and also augment short-term survival and proliferation of hHSC. Since HS may modulate BMP activity, we examined how combinations of OS-HS, BMPs and specific BMP antagonists influence PHP in umbilical cord blood (UCB). First, we confirmed by real-time quantitative RT-PCR (qRT-PCR) that UCB CD34+ and/or CD34+/CD38− cells (using linear mRNA amplification) constitutively express transcripts for BMP-4 and its inhibitor Chordin, BMP receptors BMPR-IA, BMPR-IB, BMPR-II, AcvR-II and AcvR-IIB, downstream signaling proteins SMAD-1 and -5, and target genes upregulated by BMPs including Inhibitors of DNA binding (Id) proteins 1–4, and determined their relative levels of expression. BMP-4 upregulated Id2 expression 17-fold, confirming that the BMP signaling pathway is functionally active in CD34+ cells. Next, we demonstrated that OS-HS may protect BMP-4 from its inhibitors, using Western immunoblotting of immunoprecipitated proteins to show that direct binding of the antagonist Chordin to BMP-4 is inhibited by OS-HS in a dose-dependent manner. Finally, we examined the effect of exogenous supplementation with 6 BMPs, or inhibition of endogenous BMPs by 7 antagonists, on short-term (2 wk) and long-term (5 wk) LTC-IC maintenance in UCB CD34+/CD38− cells cultured in presence of OS-HS, Flt3-ligand and thrombopoietin. Long-term LTC-IC maintenance was enhanced by BMP-4 (LTC-IC maintenance: 164 +/− 11% compared to culture without BMP-4; P=0.0001) but reduced by BMP-2 or BMP-7 (P

Description: Determining how extracellular matrix (ECM) components influence cytokine-induced growth and differentiation of cells in malignancies and other diseases is critical for understanding disease pathophysiology and for developing novel treatment strategies. Bone morphogenetic proteins (BMPs) regulate the growth, differentiation and apoptosis of cells in the brain, bone, bone marrow and diverse tissues. ECM glycosaminoglycans (GAGs) such as heparan sulfate (HS) interact with and influence the biological activity of a number of proteins including BMPs. We examined if heparin, endogenous HS in malignant cells and the structurally abnormal HS accumulated in Hurler cells influence BMP signaling and activity. First we showed using real-time quantitative RT-PCR (qRT-PCR) that the BMP signaling pathway including BMPs 2–7, BMP and activin receptors and Smad-1 and -5 are expressed by SaOS-2 human osteosarcoma cells. Western immunoblotting showed that BMP-4 induced Smad-1 phosphorylation, activation and nuclear translocation. Optimal Smad-1 activation was achieved by 25 ng/ml BMP-4 at 30–60 min, and blocked by the extracellular BMP antagonist chordin. BMP-4 also induced a concentration-dependent increase in alkaline phosphatase activity, indicative of induction of osteogenic differentiation in these malignant cells. Soluble heparin directly inhibited BMP-4 induced Smad-1 phosphorylation, and also markedly augmented the inhibitory effect of chordin. Similar effects were seen with N-desulfated, N-re-acetylated heparin but to a lesser degree than with heparin, indicating that N-sulfation of glucosamine residues in heparin/HS contributes to the effect of GAGs on BMP signaling. Inhibition of sulfation of endogenous GAGs by sodium chlorate augmented BMP-4 mediated increase in alkaline phosphatase, suggesting that endogenous sulfated GAGs themselves block BMP-4 mediated malignant cell differentiation. Because BMPs play a critical role in neurogenesis and osteogenesis, we also examined if GAGs that accumulate in Hurler syndrome impair BMP-4 signaling. Neurological dysfunction and skeletal abnormalities are among the most devastating manifestations of Hurler syndrome, an inborn metabolic disorder due to lack of lysosomal GAG-degrading α-L-iduronidase (IDUA) enzyme that leads to HS and dermatan sulfate GAG accumulation. We recently showed that HS in Hurler syndrome cells are structurally and functionally abnormal, and have impaired capability to bind and mediate FGF-2 signaling (Pan C et al. Blood2005;106:1956–64). In the present study, using Affymetrix microarrays we found that expression of the BMP signaling cascade including BMPs 1–8, -10 and -15, BMP and activin receptors, Smads 1–8, chordin and inhibitors of DNA binding (IDs) 1–4 is equivalent in normal and Hurler bone marrow derived multipotent progenitor cells. In Hurler cells, BMP-4 did induce a concentration- and time-dependent activation and nuclear translocation of Smad-1 (confocal immunofluorescent microscopy). However, BMP-4 activity was significantly enhanced following clearance of the abnormally accumulated GAGs in Hurler cells by recombinant IDUA enzyme, indicating that GAGs in Hurler cells impair BMP-4 activity. Thus, both endogenous GAGs and exogenous (soluble) heparin, via N- and O-sulfated disaccharide residues, inhibit BMP-4 activity. These findings have implications for understanding the pathobiology of diverse diseases, and for developing novel therapeutic agents that may restore BMP signaling and activity.

Description: Real-time quantitative RT-PCR (qRT-PCR) is a powerful tool for measuring or validating gene expression. Enrichment for the most primitive hematopoietic progenitors yields small cell numbers and thus small amounts of mRNA. Linear amplification (required for gene expression analysis) amplifies the 3′ end of mRNA and imposes limitations on qRT-PCR primer design that may require the use of sub-optimal primers that can form primer dimers (PDs). Indeed, using linear amplification and qRT-PCR to assay expression of 31 genes of the bone morphogenetic protein (BMP) signaling cascade in CD34+/CD38−/lin− human umbilical cord blood (UCB) progenitors, we found that 17/31 (55%) of products were false positives. To distinguish true target amplicons from PDs, we therefore used a five-step sequential strategy to initially generate a Primer Profile for each primer set: water blank and positive control dissociation curve analysis serial dilution gel electrophoresis and product sequencing, which we describe for one gene (BMP-2) that we found is variably expressed in UCB progenitors under different culture conditions. Total RNA was obtained from 5–20 x 103 UCB progenitors cultured in conditions that induced detectable BMP-2 expression (BMP-2+) and those that did not (BMP-2−), mRNA was linearly amplified (RiboAmp) and cDNA was synthesized using Invitrogen Superscript III. qRT-PCR was performed using Invitrogen SYBR Green qPCR SuperMix on an ABI 7900 HT Sequence Detection System. (1) Though primers were designed for the 3′ end of BMP-2 using the Primer 3 website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi) to identify optimal primers, an amplified product was seen in the water blank and in BMP-2− cDNA. Nevertheless, the primers also amplified the correct product in the positive control (SaOS-2 human osteosarcoma cells) and in BMP-2+ cDNA. (2) Although the genuine and the false positive products appeared similar on amplification (ΔRn vs cycle) plots, analysis of the derivative dissociation curves showed that their dissociation temperatures were distinct, being lower for the false positive PD (77°C vs 81°C). (3) Positive control and BMP-2+ cDNA yielded products with the expected one-cycle increase in cycle threshold (CT) for each 2-fold serial dilution, whereas PD product CTs did not change with serial dilution of BMP-2− cDNA. Importantly, PDs also formed at higher dilutions (≥ 1:16) of BMP-2+ cDNA, possibly because of low target mRNA abundance. (4) Agarose gel electrophoresis of the amplified products from the positive control and BMP-2+ cDNA showed the expected amplicon size of 107 bp, whereas the PD product was smaller (50 bp). (5) Finally, sequence analysis of the products (at the Univ. of Minnesota DNA Sequencing and Analysis Center) confirmed that the 107 bp product was identical with the 3′ end of human BMP-2, whereas the PD product did not yield any sequence. Similar differences were seen between genuine amplicons and PDs for several other genes examined. Thus, for each primer set, the Primer Profile provided the melting temperatures of the genuine amplicon and the PDs. For subsequent experiments, we were then able to reliably predict which product was a genuine amplicon by inspection of its melting temperature. These findings demonstrate the critical importance of using such a strategy to detect false positive qRT-PCR results due to PDs when using linear amplification of mRNA from primitive hematopoietic progenitors and other rare cell subpopulations.

Description: Mucopolysaccharidosis type I (MPS-I; Hurler syndrome) is an inborn metabolic disorder due to lack of the lysosomal glycosaminoglycan (GAG)-degrading enzyme alpha-L-iduronidase (IDUA). The resulting GAG accumulation causes progressive multi-system dysfunction and death in the first decade. We recently identified structural abnormalities in the accumulated GAGs that lead to defective heparin-binding cytokine signaling in MPS-I (Pan C et al, Blood 2005 in press: DOI 10.1182/blood-2005-02-0657), which may constitute a mechanism responsible for the diverse clinical manifestations of MPS-I and related mucopolysaccharidoses. Allogeneic hematopoietic stem cell transplantation (HSCT), if performed early in the disease, ameliorates many clinical features and extends life. However, HSCT is not available to all patients, and also does not adequately correct some of the most devastating features of MPS-I including mental retardation and skeletal deformities. Therefore, novel cellular, enzyme replacement and gene therapy approaches need to be developed for MPS-I. Such therapeutic strategies will best be tested in an immunodeficient host which is less likely to develop immune reactions to transplanted human or gene-corrected cells or their secreted IDUA enzyme. We have bred and characterized a homozygous immunodeficient MPS-I mouse (developed as a heterozygous MPS-I mouse on a NOD/SCID background by Jackson Labs) that is well suited for examining the efficacy of such therapeutic approaches. The phenotype of homozygous NOD/SCID/MPS-I mice closely mimicked the clinical features of human MPS-I including coarsening of facial features, skeletal deformities and diverse behavioral abnormalities. IDUA enzyme was completely undetectable in the liver, spleen, heart, lung, kidney and brain of homozygous animals (P