Joel Blanchard

A Small-Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog

The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

Selective conversion of fibroblasts into peripheral sensory neurons

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

Glucocorticoid Compounds Modify Smoothened Localization and Hedgehog Pathway Activity

The Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog-responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wild-type Smo, and mutant forms of Smo, SmoM2, and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.

Selective Identification of Hedgehog Pathway Antagonists by Direct Analysis of Smoothened Ciliary Translocation

Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smos actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.

Forward engineering neuronal diversity using direct reprogramming

The nervous system is comprised of a vast diversity of distinct neural cell types. Differences between neuronal subtypes drive the assembly of neuronal circuits and underlie the subtype specificity of many neurological diseases. Yet, because neurons are irreversibly post‐mitotic and not readily available from patients, it has not been feasible to study specific subtypes of human neurons in larger numbers. A powerful means to study neuronal diversity and neurological disease is to establish methods to produce desired neuronal subtypes in vitro. Traditionally this has been accomplished by treating pluripotent or neural stem cells with growth factors and morphogens that recapitulate exogenous developmental signals. These approaches often require extended periods of culture, which can limit their utility. However, more recently, it has become possible to produce neurons directly from fibroblasts using transcription factors and/or microRNAs. This technique referred to as direct reprogramming or transdifferentiation has proven to be a rapid, robust, and reproducible method to generate mature neurons of many different subtypes from multiple cell sources. Here, we highlight recent advances in generating neurons of specific subtypes using direct reprogramming and outline various scenarios in which induced neurons may be applied to studies of neuronal function and neurological disease.

Diverse reprogramming codes for neuronal identity

The transcriptional programs that establish neuronal identity evolved to produce the rich diversity of neuronal cell types that arise sequentially during development. Remarkably, transient expression of certain transcription factors can also endow non-neural cells with neuronal properties. The relationship between reprogramming factors and the transcriptional networks that produce neuronal identity and diversity remains largely unknown. Here, from a screen of 598 pairs of transcription factors, we identify 76 pairs of transcription factors that induce mouse fibroblasts to differentiate into cells with neuronal features. By comparing the transcriptomes of these induced neuronal cells (iN cells) with those of endogenous neurons, we define a ‘core’ cell-autonomous neuronal signature. The iN cells also exhibit diversity; each transcription factor pair produces iN cells with unique transcriptional patterns that can predict their pharmacological responses. By linking distinct transcription factor input ‘codes’ to defined transcriptional outputs, this study delineates cell-autonomous features of neuronal identity and diversity and expands the reprogramming toolbox to facilitate engineering of induced neurons with desired patterns of gene expression and related functional properties.A screen in which combinatorial pairs of transcription factors are exogenously expressed in fibroblasts identifies different combinations that reprogram these cells into induced neuronal cells with diverse functional properties.

Replacing reprogramming factors with antibodies selected from combinatorial antibody libraries

The reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is usually achieved by exogenous induction of transcription by factors acting in the nucleus. In contrast, during development, signaling pathways initiated at the membrane induce differentiation. The central idea of this study is to identify antibodies that can catalyze cellular de-differentiation and nuclear reprogramming by acting at the cell surface. We screen a lentiviral library encoding ∼100 million secreted and membrane-bound single-chain antibodies and identify antibodies that can replace either Sox2 and Myc (c-Myc) or Oct4 during reprogramming of mouse embryonic fibroblasts into iPSCs. We show that one Sox2-replacing antibody antagonizes the membrane-associated protein Basp1, thereby de-repressing nuclear factors WT1, Esrrb and Lin28a (Lin28) independent of Sox2. By manipulating this pathway, we identify three methods to generate iPSCs. Our results establish unbiased selection from autocrine combinatorial antibody libraries as a robust method to discover new biologics and uncover membrane-to-nucleus signaling pathways that regulate pluripotency and cell fate.

Replacing reprogramming factors with antibodies selected from autocrine antibody libraries

Signaling pathways initiated at the membrane establish and maintain cell fate during development and can be harnessed in the nucleus to generate induced pluripotent stem cells (iPSCs) from differentiated cells. Yet, the impact of extracellular signaling on reprogramming to pluripotency has not been systematically addressed. Here, we screen a lentiviral library encoding ˜100 million secreted and membrane-bound antibodies and identify multiple antibodies that can replace Sox2/c-Myc or Oct4 during reprogramming. We show that one Sox2-replacing antibody initiates reprogramming by antagonizing the membrane-associated protein Basp1, thereby inducing nuclear factors WT1 and Esrrb/Lin28 independent of Sox2. By successively manipulating this pathway we identify three new methods to generate iPSCs. This study expands current knowledge of reprogramming methods and mechanisms and establishes unbiased selection from autocrine antibody libraries as a powerful orthogonal platform to discover new biologics and pathways regulating pluripotency and cell fate.