The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.

Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia.

There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2's activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients <40 y old, we observed that nonphosphorylated CRMP2 was higher than in controls, while phosphorylated CRMP2 remained unchanged from control. In the brain, these changes were associated with dendritic structural abnormalities. The abundance of active CRMP2 with insufficient opposing inactive p-CRMP2 yielded a unique lowering of the p-CRMP2:CRMP2 ratio in SCZ patients, implying a disruption in the normal equilibrium between active and inactive CRMP2. These clinical data suggest that measuring CRMP2 and p-CRMP2 in peripheral blood might reflect intracerebral processes and suggest a rapid, minimally invasive, sensitive, and specific adjunctive diagnostic aid for early SCZ: increased CRMP2 or a decreased p-CRMP2:CRMP2 ratio may help cinch the diagnosis in a newly presenting young patient suspected of SCZ (versus such mimics as mania in bipolar disorder, where the ratio is high).

The Developmental & Molecular Requirements for Ensuring that Human Pluripotent Stem Cell-Derived Hair Follicle Bulge Stem Cells Have Acquired Competence for Hair Follicle Generation Following Transplantation.

When using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, we found it best to emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermogenesis. Viewing hiPSCs as a model of the epiblast, we exploited insights from mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage in order to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, we show that hiPSC derivatives should co-express, just prior to transplantation, the following combination of markers: integrins α6 and ß1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation is not possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14+ keratin 5+ CD200- keratinocytes (Day 25), HF generation is equally impossible. We make the developmental case for transplanting at Day 16-18 of differentiation-the point at which the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.

A Tool for Accurate Stoichiometric Composition of Cryopreservative Media for Fetal and Induced Pluripotent Stem Cell-Derived Human Neural Stem Cells.

Fetal human neural stem cells (fhNSC) are of considerable interest as potential regenerative therapies for neuronal or glial degeneration or destruction resulting from genetic abnormalities, disease, or injury. Realization of this potential requires securing a supply of cells sufficient to meet the needs of transplantation, which are often tens to hundreds of millions of cells per dose. This challenge necessitates the establishment of safe and efficient cell banking protocols. Cryopreservation, involving the slow freezing or vitrification of cells, enables storage of fhNSC for prolonged periods, while maintaining their viability and multipotency required for clinical use. To optimize cryopreservation of fhNSC, attention has become focused on the composition of the medium used to effect cryopreservation by slow freezing/vitrification-i.e., the cryopreservative medium. The cryopreservative medium is typically specified as a dilution of a concentrated cryoprotectant, such as dimethylsulfoxide or glycerol, in cell culture medium that is often combined with serum or another source of necessary growth factors. The present work is devoted to a computational tool for determining the composition of a cryopreservative medium that can be combined with dissociated fhNSC resuspended in a certain volume of culture medium to achieve the criterion of stoichiometric dilution of cryoprotectant favorable to cell viability in the final mixture of cryopreservative medium and cells. © 2021 Wiley Periodicals LLC. Basic Protocol: Culture and passage of fhNSC, counting of enzymatically dissociated fhNSC, and quantitative formulation of cryomedium Alternate Protocol: Procedure when cell medium is not added to the cryomedium.

Meningeal Multipotent Cells: A Hidden Target for CNS Repair?

Traditionally, the primary role of the meninges is thought to be structural, i.e., to act as a surrounding membrane that contains and cushions the brain with cerebrospinal fluid. During development, the meninges is formed by both mesenchymal and neural crest cells. There is now emerging evidence that subsets of undifferentiated stem cells might persist in the adult meninges. In this mini-review, we survey representative studies of brain-meningeal interactions and discuss the hypothesis that the meninges are not just protective membranes, but instead contain multiplex stem cell subsets that may contribute to central nervous system (CNS) homeostasis. Further investigations into meningeal multipotent cells may reveal a "hidden" target for promoting neurovascular remodeling and repair after CNS injury and disease.

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFRα). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

Strategies and Progress in CXCR4-Targeted Anti-Human Immunodeficiency Virus (HIV) Therapeutic Development.

The acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV), has been a global public health challenge for several decades. The majority of HIV infection is caused by the human immunodeficiency virus type 1 (HIV-1), which enters and infects a host cell via the cell surface proteins of CD4 as the primary receptor, and chemokine receptors CXCR4 or CCR5 as the coreceptor-then undergoing replication using the cell's intracellular machinery. Whereas many drugs targeting CCR5-mediated entry or HIV-1 replication via reverse transcriptase or proteases have long been used clinically, agents targeting CXCR4 are yet to be advanced to clinical application. Here in this review we highlight some of the strategies for and progress made in the discovery of novel small molecules, peptides, and larger molecules that target CXCR4, and their future prospects for translation into the clinic as a new class of anti-HIV therapeutics.

Valproate reverses mania-like behaviors in mice via preferential targeting of HDAC2.

Valproate (VPA) has been used in the treatment of bipolar disorder since the 1990s. However, the therapeutic targets of VPA have remained elusive. Here we employ a preclinical model to identify the therapeutic targets of VPA. We find compounds that inhibit histone deacetylase proteins (HDACs) are effective in normalizing manic-like behavior, and that class I HDACs (e.g., HDAC1 and HDAC2) are most important in this response. Using an RNAi approach, we find that HDAC2, but not HDAC1, inhibition in the ventral tegmental area (VTA) is sufficient to normalize behavior. Furthermore, HDAC2 overexpression in the VTA prevents the actions of VPA. We used RNA sequencing in both mice and human induced pluripotent stem cells (iPSCs) derived from bipolar patients to further identify important molecular targets. Together, these studies identify HDAC2 and downstream targets for the development of novel therapeutics for bipolar mania.

Countermeasures for Preventing and Treating Opioid Overdose.

The only medication available currently to prevent and treat opioid overdose (naloxone) was approved by the US Food and Drug Administration (FDA) nearly 50 years ago. Because of its pharmacokinetic and pharmacodynamic properties, naloxone has limited utility under some conditions and would not be effective to counteract mass casualties involving large-scale deployment of weaponized synthetic opioids. To address shortcomings of current medical countermeasures for opioid toxicity, a trans-agency scientific meeting was convened by the US National Institute of Allergy and Infectious Diseases/National Institutes of Health (NIAID/NIH) on August 6 and 7, 2019, to explore emerging alternative approaches for treating opioid overdose in the event of weaponization of synthetic opioids. The meeting was initiated by the Chemical Countermeasures Research Program (CCRP), was organized by NIAID, and was a collaboration with the National Institute on Drug Abuse/NIH (NIDA/NIH), the FDA, the Defense Threat Reduction Agency (DTRA), and the Biomedical Advanced Research and Development Authority (BARDA). This paper provides an overview of several presentations at that meeting that discussed emerging new approaches for treating opioid overdose, including the following: (1) intranasal nalmefene, a competitive, reversible opioid receptor antagonist with a longer duration of action than naloxone; (2) methocinnamox, a novel opioid receptor antagonist; (3) covalent naloxone nanoparticles; (4) serotonin (5-HT)1A receptor agonists; (5) fentanyl-binding cyclodextrin scaffolds; (6) detoxifying biomimetic "nanosponge" decoy receptors; and (7) antibody-based strategies. These approaches could also be applied to treat opioid use disorder.

Chemical mutagenesis of a GPCR ligand: Detoxifying "inflammo-attraction" to direct therapeutic stem cell migration.

A transplanted stem cell's engagement with a pathologic niche is the first step in its restoring homeostasis to that site. Inflammatory chemokines are constitutively produced in such a niche; their binding to receptors on the stem cell helps direct that cell's "pathotropism." Neural stem cells (NSCs), which express CXCR4, migrate to sites of CNS injury or degeneration in part because astrocytes and vasculature produce the inflammatory chemokine CXCL12. Binding of CXCL12 to CXCR4 (a G protein-coupled receptor, GPCR) triggers repair processes within the NSC. Although a tool directing NSCs to where needed has been long-sought, one would not inject this chemokine in vivo because undesirable inflammation also follows CXCL12-CXCR4 coupling. Alternatively, we chemically "mutated" CXCL12, creating a CXCR4 agonist that contained a strong pure binding motif linked to a signaling motif devoid of sequences responsible for synthetic functions. This synthetic dual-moity CXCR4 agonist not only elicited more extensive and persistent human NSC migration and distribution than did native CXCL 12, but induced no host inflammation (or other adverse effects); rather, there was predominantly reparative gene expression. When co-administered with transplanted human induced pluripotent stem cell-derived hNSCs in a mouse model of a prototypical neurodegenerative disease, the agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cerebral cortex (including as electrophysiologically-active cortical neurons) where their secreted cross-corrective enzyme mediated a therapeutic impact unachieved by cells alone. Such a "designer" cytokine receptor-agonist peptide illustrates that treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., "inflammo-attraction").

Nna1 gene deficiency triggers Purkinje neuron death by tubulin hyperglutamylation and ER dysfunction.

Posttranslational glutamylation/deglutamylation balance in tubulins influences dendritic maturation and neuronal survival of cerebellar Purkinje neurons (PNs). PNs and some additional neuronal types degenerate in several spontaneous, independently occurring Purkinje cell degeneration (pcd) mice featuring mutant neuronal nuclear protein induced by axotomy (Nna1), a deglutamylase gene. This defective deglutamylase allows glutamylases to form hyperglutamylated tubulins. In pcd, all PNs die during postnatal "adolescence." Neurons in some additional brain regions also die, mostly later than PNs. We show in laser capture microdissected single PNs, in cerebellar granule cell neuronal clusters, and in dissected hippocampus and substantia nigra that deglutamase mRNA and protein were virtually absent before pcd PNs degenerated, whereas glutaminase mRNA and protein remained normal. Hyperglutamylated microtubules and dimeric tubulins accumulated in pcd PNs and were involved in pcd PN death by glutamylase/deglutamylase imbalance. Importantly, treatment with a microtubule depolymerizer corrected the glutamylation/deglutamylation ratio, increasing PN survival. Further, before onset of neuronal death, pcd PNs displayed prominent basal polylisosomal masses rich in ER. We propose a "seesaw" metamorphic model summarizing mutant Nna1-induced tubulin hyperglutamylation, the pcd's PN phenotype, and report that the neuronal disorder involved ER stress, unfolded protein response, and protein synthesis inhibition preceding PN death by apoptosis/necroptosis.

Deriving Keratinocyte Progenitor Cells and Keratinocytes from Human-Induced Pluripotent Stem Cells.

Skin or hair loss (alopecia) may occur due to a wide variety of causes ranging from trauma to pathological processes including acquired or congenital causes. It would be ideal to replace them with immunologically compatible cells to avoid potentially exacerbating the condition. Deriving the replacement cells from human-induced pluripotent stem cells (hiPSCs) allows for sufficient scale up and using hiPSCs as the choice of human pluripotent stem cells (hPSC) will ensure immunocompatibility. Here we offer a protocol for differentiating hiPSCs into keratinocyte progenitor cells (KPC) and keratinocytes employing all-trans retinoic acid (ATRA) and L-ascorbic acid, (L-AA), bone morphogenic protein-4 (BMP4), and epidermal growth factor (EGF). We observed that the hiPSC-derived KPCs express the same panel of markers as primary hair follicle bulge stem cells (HFBSCs), including CD200, integrin α-6 (ITGA6), integrin ß-1 (ITGB1), the transcription factor P63, keratin 15 (KRT15), and keratin 19 (KRT19). If permitted to differentiate further, the hiPSC-derived KPC lose CD200 expression and rather come to express keratin 14 (KRT14) indicating emergence of more mature terminally-differentiated keratinocytes. The HFBSCs are transplantable for hair follicle (HF) restoration, and the keratinocytes may be transplantable for therapy for large burns or ulcers. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Reprogramming of normal human skin fibroblasts into normal hiPSCs using episomal DNA cocktail Basic Protocol 2: Differentiation of hiPSCs into KPCs and keratinocytes Alternate Protocol 2: EBS formation protocol using AggreWell™ plates (Antonchuk, 2013) Support Protocol 1: Passage hiPSC-KPC Support Protocol 2: Immunocytochemistry (ICC) Support Protocol 3: Immunofluorescence staining of cells for flow cytometry (FC).

"Reprogram Enablement" as an Assay for Identifying Early Oncogenic Pathways by Their Ability to Allow Neoplastic Cells to Reacquire an Epiblast State.

One approach to understanding how tissue-specific cancers emerge is to determine the requirements for "reprograming" such neoplastic cells back to their developmentally normal primordial pre-malignant epiblast-like pluripotent state and then scrutinizing their spontaneous reconversion to a neoplasm, perhaps rendering salient the earliest pivotal oncogenic pathway(s) (before other aberrations accumulate in the adult tumor). For the prototypical malignancy anaplastic thyroid carcinoma (ATC), we found that tonic RAS reduction was obligatory for reprogramming cancer cells to a normal epiblast-emulating cells, confirmed by changes in their transcriptomic and epigenetic profiles, loss of neoplastic behavior, and ability to derive normal somatic cells from their "epiblast organoids." Without such suppression, ATCs re-emerged from the clones. Hence, for ATC, RAS inhibition was its "reprogram enablement" (RE) factor. Each cancer likely has its own RE factor; identifying it may illuminate pre-malignant risk markers, better classifications, therapeutic targets, and tissue-specification of a previously pluripotent, now neoplastic, cell.

Generation of Complete Multi-Cell Type Lung Organoids From Human Embryonic and Patient-Specific Induced Pluripotent Stem Cells for Infectious Disease Modeling and Therapeutics Validation.

The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.

A Biomarker for Predicting Responsiveness to Stem Cell Therapy Based on Mechanism-of-Action: Evidence from Cerebral Injury.

To date, no stem cell therapy has been directed to specific recipients-and, conversely, withheld from others-based on a clinical or molecular profile congruent with that cell's therapeutic mechanism-of-action (MOA) for that condition. We address this challenge preclinically with a prototypical scenario: human neural stem cells (hNSCs) against perinatal/neonatal cerebral hypoxic-ischemic injury (HII). We demonstrate that a clinically translatable magnetic resonance imaging (MRI) algorithm, hierarchical region splitting, provides a rigorous, expeditious, prospective, noninvasive "biomarker" for identifying subjects with lesions bearing a molecular profile indicative of responsiveness to hNSCs' neuroprotective MOA. Implanted hNSCs improve lesional, motor, and/or cognitive outcomes only when there is an MRI-measurable penumbra that can be forestalled from evolving into necrotic core; the core never improves. Unlike the core, a penumbra is characterized by a molecular profile associated with salvageability. Hence, only lesions characterized by penumbral > core volumes should be treated with cells, making such measurements arguably a regenerative medicine selection biomarker.

Discovery of suppressors of CRMP2 phosphorylation reveals compounds that mimic the behavioral effects of lithium on amphetamine-induced hyperlocomotion.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3ß, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/ß with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/ß inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3ß kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

The Evolution of Stem Cells, Disease Modeling, and Drug Discovery for Neurological Disorders.

Human neurological disorders are among the most challenging areas of translational research. The difficulty of acquiring human neural samples or specific representative animal models has necessitated a multifaceted approach to understanding disease pathology and drug discovery. The dedifferentiation of somatic cells to human induced pluripotent stem cells (hiPSCs) for the generation of neural derivatives has broadened the capability of biomedical research to study human cell types in neurological disorders. The initial zeal for the potential of hiPSCs for immediate biomedical breakthroughs has evolved to more reasonable expectations. Over the past decade, hiPSC technology has demonstrated the capacity to successfully establish "disease in a dish" models of complex neurological disorders and to identify possible novel therapeutics. However, as hiPSCs are used more broadly, an increased understanding of the limitations of hiPSC studies is becoming more evident. In this study, we review the challenges of studying neurological disorders, the current limitations of stem cell-based disease modeling, and the degrees to which hiPSC studies to date have demonstrated the capacity to fill essential gaps in neurological research.

Stem Cell Composition of Umbilical Cord Blood Following Milking Compared with Delayed Clamping of the Cord Appears Better Suited for Promoting Hematopoiesis.

In comparing placental transfusion strategies, blood obtained from an umbilical cord that has been "milked" vs one in which clamping was simply delayed contains mesenchymal stromal cells in addition to solely hematopoietic stem cells, a composition more favorable for hematopoiesis, as suggested by its superior rescue of lethally irradiated bone marrow-depleted mice.

Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

Identifying cellular programs that drive cancers to be stem-like and treatment resistant is critical to improving outcomes in patients. Here, we demonstrate that constitutive extracellular signal-regulated kinase 1/2 (ERK1/2) activation sustains a stem-like state in glioblastoma (GBM), the most common primary malignant brain tumor. Pharmacological inhibition of ERK1/2 activation restores neurogenesis during murine astrocytoma formation, inducing neuronal differentiation in tumorspheres. Constitutive ERK1/2 activation globally regulates miRNA expression in murine and human GBMs, while neuronal differentiation of GBM tumorspheres following the inhibition of ERK1/2 activation requires the functional expression of miR-124 and the depletion of its target gene SOX9. Overexpression of miR124 depletes SOX9 in vivo and promotes a stem-like-to-neuronal transition, with reduced tumorigenicity and increased radiation sensitivity. Providing a rationale for reports demonstrating miR-124-induced abrogation of GBM aggressiveness, we conclude that reversal of an ERK1/2-miR-124-SOX9 axis induces a neuronal phenotype and that enforcing neuronal differentiation represents a therapeutic strategy to improve outcomes in GBM.

Modeling Complex Neurological Diseases with Stem Cells: A Study of Bipolar Disorder.

The pathogenesis of bipolar disorder (BPD) is unknown. Using human-induced pluripotent stem cells (hiPSCs) to unravel pathological mechanisms in polygenic diseases is challenging, with few successful studies to date. However, hiPSCs from BPD patients responsive to lithium have offered unique opportunities to discern lithium's mechanism of action and hence gain insight into BPD pathology. By profiling the proteomics of BPD-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). The "set point" for the ratio of pCRMP2:CRMP2 is elevated uniquely in hiPSC-derived neurons from lithium responsive (Li-R) BPD patients, but not other psychiatric and neurological disorders. Utilizing neurons differentiated from human patient stem cells as an in vitro platform, we were able to elucidate the mechanism driving the pathogenesis and pathophysiology of lithium-responsive BPD, heretofore unknown. Importantly, the findings in culture were validated in human postmortem material as well as in animal models of BPD behavior. These data suggest that the "lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in dendritic spines, leading to modulated neural networks that may underlie Li-R BPD pathogenesis. This chapter reviews the methodology of leveraging a functional agent, lithium, to identify unknown pathophysiological pathways with hiPSCs and how to translate this disease modeling approach to other neurological disorders.