The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons

Abstract

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.

Introduction

Biochemical cues within the stem cell niche that instruct cell fate decisions are often incorporated into larger structures, for example via self-assembly or by immobilization to the extracellular matrix. Understanding the importance of this nanoscale spatial organization in controlling cell behavior both advances our basic knowledge of stem cell and developmental biology and enables applications in tissue engineering and regenerative medicine. Multivalent interactions – where multiple ligands on one entity bind multiple receptors on another – are one such class of nanoscale organization that naturally occur in many biological processes ranging from growth factor and morphogen signaling to the attachment of a virus to a cell surface [1]. Multivalent ligands are often collectively more potent than corresponding monovalent interactions due to an enhanced ability to occupy and/or cluster their receptors [2], [3], [4]. Engineered biomaterials are increasingly being employed to design systems that emulate important biological features of natural niches [5], [6], and synthetic biomimetic multivalent ligands may be more potent than monovalent ligands in regulating cell fate decisions, such as stem cell differentiation.

Shh, a potent morphogen that specifies cell fate choices in tissues throughout the developing embryo is one factor that has been suggested to function as a multimeric form [7], [8], [9]. Within the neural tube – the germinal origin of the central nervous system (CNS) – a gradient of Shh initially emanating from ventral mesenchyme tissue and subsequently the floor plate cells within the tube patterns differentiation of ventral progenitor domains in a concentration dependent manner [10]. The resulting ventral progenitors give rise to many neuronal cell types along the neuraxis including midbrain dopaminergic (mDA) [11] and GABA producing inhibitory neurons [12], which undergo degeneration in Parkinson's disease (PD) [13] or are impaired in epileptic disorders [14], [15], [16], respectively. Efforts to develop cell replacement therapies for these intractable neurodegenerative diseases routinely manipulate Shh signaling to ventralize neurally differentiating human pluripotent stem cells (hPSCs) and thereby generate mDA [17] or GABAergic [15] progenitors.

Small molecule agonists of Shh signaling have been used to induce the dopaminergic and GABAergic differentiation of hPSCs. Recombinant Shh, however, is 1–2 orders of magnitude more potent on a molar basis in patterning neural cell fate [18], [19], and when used in combination with saturating levels of Shh agonist purmorphamine, recombinant Shh can further increase the efficacy of lineage-specific neuronal differentiation protocols [17]. Therefore, it appears that activation of Shh signaling with the protein ligand, which binds to the cell membrane receptor Patched, offers potential advantages compared to small molecule agonists, which regulate the downstream effector Smoothened (SMO).

Naturally produced Shh is covalently modified by cholesterol and palmitate [20], [21]. These lipid moieties were initially believed to tether the protein to the cellular plasma membrane, yet in the neural tube Shh secreted from the notochord and floor plate act in long range to organize the pattern of ventral neurogenesis [7], [22]. Shh's long-range signaling effects observed during organismal development have recently been attributed to its nanoscale clustering and multimerization within the secreting cell's membrane prior to release as a diffusible and multivalent molecule [7], [8], [9], [23], [24]. Furthermore, recent observations that Shh can be assembled into a soluble multimeric protein complex with a hydrophobic core of lipids help explain this transport [25]. The resulting multimeric form of Shh is also reported in vitro to be even more potent than monovalent recombinant Shh [7], [24]; however, Shh multimerization and secretion rely on complex mammalian posttranslational modifications and secretory mechanisms that are not fully understood [26] and that render the production of natural, multivalent Shh for regenerative medicine applications problematic. We previously demonstrated that a bio-inspired, multivalent, conjugate form of Shh was more active in a murine fibroblast bioassay [27], raising the possibility that multivalent Shh bioconjugates may potentially serve as valuable materials to more effectively direct the differentiation of hPSCs into therapeutically valuable cell types compared to recombinant Shh or small molecule agonists of Shh signaling.

In this work we generated multivalent Shh with defined spatial distribution by conjugating recombinant Shh to linear Hyaluronic acid (HyA) polymers at various stoichiometric ratios and investigated the putative role of multivalency in Shh signaling during neuronal differentiation of hPSCs. We evaluated whether biomimetic Shh conjugates can be used as a bioactive material to enhance ventralization of neural progenitors and lineage commitment to therapeutically relevant neuronal phenotypes including mDA and GABAergic neurons in direct comparison with equimolar amounts of monomeric recombinant Shh, and ten-fold higher levels of Shh pathway small molecule agonist (SAG).