Heparan sulfate regulation of progenitor cell fate

Currently there is an intense effort being made to elucidate the factors that control stem and progenitor cell fate. Developments in our understanding of the FGF/FGFR pathway and its role as an effector of stem cell pluripotency have heightened expectations that a therapeutic use for stem cells will move from a possibility to a probability. Mounting evidence is revealing the molecular mechanisms by which fibroblast growth factor (FGF) signaling, together with a large number of other growth and adhesive factors, is controlled by the extracellular sugar, heparan sulfate (HS). What has resulted is a novel means of augmenting and thus regulating the growth factor control of stem and progenitor cell fate. Here, we review the numerous bioactivities of HS, and the development of strategies to implement HS-induced control of cell fate decisions.     

Heparan Sulfate Biosynthesis Enzymes in Embryonic Stem Cell Biology

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and can give rise to all cell types in the body. The fate of ES cells depends on the signals they receive from their surrounding environment, which either promote self-renewal or initiate differentiation. Heparan sulfate proteoglycans are macromolecules found on the cell surface and in the extracellular matrix. Acting as low-affinity receptors on the cell surface, heparan sulfate (HS) side chains modulate the functions of numerous growth factors and morphogens, having wide impact on the extracellular information received by cells. ES cells lacking HS fail to differentiate but can be induced to do so by adding heparin. ES cells defective in various components of the HS biosynthesis machinery, thus expressing differently flawed HS, exhibit lineage-specific effects. Here we discuss recent studies on the biological functions of HS in ES cell developmental processes. Since ES cells have significant potential applications in tissue/cell engineering for cell replacement therapies, understanding the functional mechanisms of HS in manipulating ES cell growth in vitro is of utmost importance, if the stem cell regenerative medicine from scientific fiction ever will be made real.     

A role for extra macrochaetae downstream of Notch in follicle cell differentiation

The Drosophila ovary provides a model system for studying the mechanisms that regulate the differentiation of somatic stem cells into specific cell types. Ovarian somatic stem cells produce follicle cells, which undergo a binary choice during early differentiation. They can become either epithelial cells that surround the germline to form an egg chamber ('main body cells') or a specialized cell lineage found at the poles of egg chambers. This lineage goes on to make two cell types: polar cells and stalk cells. To better understand how this choice is made, we carried out a screen for genes that affect follicle cell fate specification or differentiation. We identified extra macrochaetae (emc), which encodes a helix-loop-helix protein, as a downstream effector of Notch signaling in the ovary. EMC is expressed in proliferating cells in the germarium, as well as in the main body follicle cells. EMC expression in the main body cells is Notch dependent, and emc mutant cells located on the main body failed to differentiate. EMC expression is reduced in the precursors of the polar and stalk cells, and overexpression of EMC caused dramatic egg chamber fusions, indicating that EMC is a negative regulator of polar and/or stalk cells. EMC and Notch were both required in the main body cells for expression of Eyes Absent (EYA), a negative regulator of polar and stalk cell fate. We propose that EMC functions downstream of Notch and upstream of EYA to regulate main body cell fate specification and differentiation.     

Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells

Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.     

Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment

Hematopoiesis is maintained by stem cells (HSCs) that undergo fate decisions by integrating intrinsic and extrinsic signals, with the latter derived from the bone marrow (BM) microenvironment. Cell-cycle regulation can modulate stem cell fate, but it is unknown whether this represents an intrinsic or extrinsic effector of fate decisions. We have investigated the role of the retinoblastoma protein (RB), a central regulator of the cell cycle, in hematopoiesis. Widespread inactivation of RB in the murine hematopoietic system resulted in profound myeloproliferation. HSCs were lost from the BM due to mobilization to extramedullary sites and differentiation. This phenotype was not intrinsic to HSCs, but, rather, was the consequence of an RB-dependent interaction between myeloid-derived cells and the microenvironment. These findings demonstrate that myeloproliferation may result from perturbed interactions between hematopoietic cells and the niche. Therefore, RB extrinsically regulates HSCs by maintaining the capacity of the BM to support normal hematopoiesis and HSCs.     

Sending the right signal: Notch and stem cells

Background

Notch signaling plays a critical role in multiple developmental programs and not surprisingly, the Notch pathway has also been implicated in the regulation of many adult stem cells, such as those in the intestine, skin, lungs, hematopoietic system, and muscle.

Scope of review

In this review, we will first describe molecular mechanisms of Notch component modulation including recent advances in this field and introduce the fundamental principles of Notch signaling controlling cell fate decisions. We will then illustrate its important and varied functions in major stem cell model systems including: Drosophila and mammalian intestinal stem cells and mammalian skin, lung, hematopoietic and muscle stem cells.

Major conclusions

The Notch receptor and its ligands are controlled by endocytic processes that regulate activation, turnover, and recycling. Glycosylation of the Notch extracellular domain has important modulatory functions on interactions with ligands and on proper receptor activity. Notch can mediate cell fate decisions including proliferation, lineage commitment, and terminal differentiation in many adult stem cell types. Certain cell fate decisions can have precise requirements for levels of Notch signaling controlled through modulatory regulation.

General significance

We describe the current state of knowledge of how the Notch receptor is controlled through its interaction with ligands and how this is regulated by associated factors. The functional consequences of Notch receptor activation on cell fate decisions are discussed. We illustrate the importance of Notch's role in cell fate decisions in adult stem cells using examples from the intestine, skin, lung, blood, and muscle. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Auxin gradients trigger de novo formation of stem cells during somatic embryogenesis

Single or a group of somatic cells could give rise to the whole plant, which require hormones, or plant growth regulators. Although many studies have been done during past years, how hormones specify cell fate during in vitro organogenesis is still unknown. To uncover this mechanism, Arabidopsis somatic embryogenesis has been recognized as a model for studying in vitro plant organogenesis. In this paper, we showed that establishment of auxin gradients within embryonic callus is essential for inducing stem cell formation via PIN1 regulation. This study sheds new light on how hormone regulates stem cell formation during in vitro organogenesis.Key words: auxin gradients, PIN proteins, stem cell, somatic embryogenesis     

SNW1 is a critical regulator of spatial BMP activity, neural plate border formation, and neural crest specification in vertebrate embryos

Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification.     

Targeting of CD44 eradicates human acute myeloid leukemic stem cells

The long-term survival of patients with acute myeloid leukemia (AML) is dismally poor. A permanent cure of AML requires elimination of leukemic stem cells (LSCs), the only cell type capable of initiating and maintaining the leukemic clonal hierarchy. We report a therapeutic approach using an activating monoclonal antibody directed to the adhesion molecule CD44. In vivo administration of this antibody to nonobese diabetic-severe combined immune-deficient mice transplanted with human AML markedly reduced leukemic repopulation. Absence of leukemia in serially transplanted mice demonstrated that AML LSCs are directly targeted. Mechanisms underlying this eradication included interference with transport to stem cell-supportive microenvironmental niches and alteration of AML-LSC fate, identifying CD44 as a key regulator of AML LSCs. The finding that AML LSCs require interaction with a niche to maintain their stem cell properties provides a therapeutic strategy to eliminate quiescent AML LSCs and may be applicable to other types of cancer stem cells.     

Odd-skipped maintains prohemocyte potency and blocks blood cell development in Drosophila

Studies using Drosophila have contributed significantly to our understanding of regulatory mechanisms that control stem cell fate choice. The Drosophila blood cell progenitor or prohemocyte shares important characteristics with mammalian hematopoietic stem cells, including quiescence, niche dependence, and the capacity to form all three fly blood cell types. This report extends our understanding of prohemocyte fate choice by showing that the zinc-finger protein Odd-skipped promotes multipotency and blocks differentiation. Odd-skipped was expressed in prohemocytes and downregulated in terminally differentiated plasmatocytes. Furthermore, Odd-skipped maintained the prohemocyte population and blocked differentiation of plasmatocytes and lamellocytes but not crystal cells. A previous study showed that Odd-skipped expression is downregulated by Decapentaplegic signaling. This report provides a functional basis for this regulator/target pair by suggesting that Decapentaplegic signaling limits Odd-skipped expression to promote prohemocyte differentiation. Overall, these studies are the basis for a gene regulatory model of prohemocyte cell fate choice.     

Cytoskeletal polarity mediates localized induction of the heart progenitor lineage

Cells must make appropriate fate decisions within a complex and dynamic environment. In vitro studies indicate that the cytoskeleton acts as an integrative platform for this environmental input. External signals regulate cytoskeletal dynamics and the cytoskeleton reciprocally modulates signal transduction. However, in vivo studies linking cytoskeleton/signalling interactions to embryonic cell fate specification remain limited. Here we show that the cytoskeleton modulates heart progenitor cell fate. Our studies focus on differential induction of heart fate in the basal chordate Ciona intestinalis. We have found that differential induction does not simply reflect differential exposure to the inductive signal. Instead, pre-cardiac cells employ polarized, invasive protrusions to localize their response to an ungraded signal. Through targeted manipulation of the cytoskeletal regulator CDC42, we are able to depolarize protrusive activity and generate uniform heart progenitor fate specification. Furthermore, we are able to restore differential induction by repolarizing protrusive activity. These findings illustrate how bi-directional interactions between intercellular signalling and the cytoskeleton can influence embryonic development. In particular, these studies highlight the potential for dynamic cytoskeletal changes to refine cell fate specification in response to crude signal gradients.     

Heparan sulfate proteoglycans as key regulators of the mesenchymal niche of hematopoietic stem cells

The complex microenvironment that surrounds hematopoietic stem cells (HSCs) in the bone marrow niche involves different coordinated signaling pathways. The stem cells establish permanent interactions with distinct cell types such as mesenchymal stromal cells, osteoblasts, osteoclasts or endothelial cells and with secreted regulators such as growth factors, cytokines, chemokines and their receptors. These interactions are mediated through adhesion to extracellular matrix compounds also. All these signaling pathways are important for stem cell fates such as self-renewal, proliferation or differentiation, homing and mobilization, as well as for remodeling of the niche. Among these complex molecular cues, this review focuses on heparan sulfate (HS) structures and functions and on the role of enzymes involved in their biosynthesis and turnover. HS associated to core protein, constitute the superfamily of heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix of all tissues. The key regulatory effects of major medullar HSPGs are described, focusing on their roles in the interactions between hematopoietic stem cells and their endosteal niche, and on their ability to interact with Heparin Binding Proteins (HBPs). Finally, according to the relevance of HS moieties effects on this complex medullar niche, we describe recent data that identify HS mimetics or sulfated HS signatures as new glycanic tools and targets, respectively, for hematopoietic and mesenchymal stem cell based therapeutic applications.     

Blood Vessels Pattern Heparan Sulfate Gradients between Their Apical and Basolateral Aspects

A hallmark of immune cell trafficking is directional guidance via gradients of soluble or surface bound chemokines. Vascular endothelial cells produce, transport and deposit either their own chemokines or chemokines produced by the underlying stroma. Endothelial heparan sulfate (HS) was suggested to be a critical scaffold for these chemokine pools, but it is unclear how steep chemokine gradients are sustained between the lumenal and ablumenal aspects of blood vessels. Addressing this question by semi-quantitative immunostaining of HS moieties around blood vessels with a pan anti-HS IgM mAb, we found a striking HS enrichment in the basal lamina of resting and inflamed post capillary skin venules, as well as in high endothelial venules (HEVs) of lymph nodes. Staining of skin vessels with a glycocalyx probe further suggested that their lumenal glycocalyx contains much lower HS density than their basolateral extracellular matrix (ECM). This polarized HS pattern was observed also in isolated resting and inflamed microvascular dermal cells. Notably, progressive skin inflammation resulted in massive ECM deposition and in further HS enrichment around skin post capillary venules and their associated pericytes. Inflammation-dependent HS enrichment was not compromised in mice deficient in the main HS degrading enzyme, heparanase. Our results suggest that the blood vasculature patterns steep gradients of HS scaffolds between their lumenal and basolateral endothelial aspects, and that inflammatory processes can further enrich the HS content nearby inflamed vessels. We propose that chemokine gradients between the lumenal and ablumenal sides of vessels could be favored by these sharp HS scaffold gradients.