Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development-Current State-of-the-Art and Future Perspectives.

The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.

SARS-CoV-2 promotes microglial synapse elimination in human brain organoids.

Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection initiate neuronal cell death and cause a loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synapse engulfment. To a large extent, SARS-CoV-2 exposed microglia adopt a transcriptomic profile overlapping with neurodegenerative disorders that display an early synapse loss as well as an increased incident risk after a SARS-CoV-2 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from COVID-19.

Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons.

Cerebrospinal fluid concentration of complement component 4A is increased in first episode schizophrenia.

Postsynaptic density is reduced in schizophrenia, and risk variants increasing complement component 4A (C4A) gene expression are linked to excessive synapse elimination. In two independent cohorts, we show that cerebrospinal fluid (CSF) C4A concentration is elevated in patients with first-episode psychosis (FEP) who develop schizophrenia (FEP-SCZ: median 0.41 fmol/ul [CI = 0.34-0.45], FEP-non-SCZ: median 0.29 fmol/ul [CI = 0.22-0.35], healthy controls: median 0.28 [CI = 0.24-0.33]). We show that the CSF elevation of C4A in FEP-SCZ exceeds what can be expected from genetic risk variance in the C4 locus, and in patient-derived cellular models we identify a mechanism dependent on the disease-associated cytokines interleukin (IL)-1beta and IL-6 to selectively increase neuronal C4A mRNA expression. In patient-derived CSF, we confirm that IL-1beta correlates with C4A controlled for genetically predicted C4A RNA expression (r = 0.39; CI: 0.01-0.68). These results suggest a role of C4A in early schizophrenia pathophysiology.

Involvement of sphingosine-1-phosphate in glutamate secretion in hippocampal neurons.

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P(1) receptor at the puncta was activated during depolarization and that depolarization-induced S1P(1) receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na(+)-channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.

The S1P2 receptor negatively regulates platelet-derived growth factor-induced motility and proliferation.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G protein-coupled receptors, named S1P(1) to S1P(5). In this study, we found that cross-communication between platelet-derived growth factor receptor and S1P(2) serves as a negative damper of PDGF functions. Deletion of the S1P(2) receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P(1) and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P(2) deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P(2)-null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P(2) reduced DNA synthesis and expression of SphK1. Thus, S1P(2) serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.

Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors.

BACKGROUND: Transplantation of pancreatic ß cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)-PDX1 and NKX6.1-has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. METHODS: synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. RESULTS: Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. CONCLUSIONS: In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.

Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells.

Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.

Amyotrophic lateral sclerosis model derived from human embryonic stem cells overexpressing mutant superoxide dismutase 1.

The generation of amyotrophic lateral sclerosis (ALS) disease models is an important subject for investigating disease mechanisms and pharmaceutical applications. In transgenic mice, expression of a mutant form of superoxide dismutase 1 (SOD1) can lead to the development of ALS that closely mimics the familial type of ALS (FALS). Although SOD1 mutant mice show phenotypes similar to FALS, dissimilar drug responses and size differences limit their usefulness to study the disease mechanism(s) and identify potential therapeutic compounds. Development of an in vitro model system for ALS is expected to help in obtaining novel insights into disease mechanisms and discovery of therapeutics. We report the establishment of an in vitro FALS model from human embryonic stem cells overexpressing either a wild-type (WT) or a mutant SOD1 (G93A) gene and the evaluation of the phenotypes and survival of the spinal motor neurons (sMNs), which are the neurons affected in ALS patients. The in vitro FALS model that we developed mimics the in vivo human ALS disease in terms of the following: (a) selective degeneration of sMNs expressing the G93A SOD1 but not those expressing the WT gene; (b) susceptibility of G93A SOD1-derived sMNs to form ubiquitinated inclusions; (c) astrocyte-derived factor(s) in the selective degeneration of G93A SOD1 sMNs; and (d) cell-autonomous, as well as non-cell-autonomous, dependent sMN degeneration. Thus, this model is expected to help unravel the disease mechanisms involved in the development of FALS and also lead to potential drug discoveries based on the prevention of neurodegeneration.

Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2.

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.

Novel role of sphingosine kinase 1 as a mediator of neurotrophin-3 action in oligodendrocyte progenitors.

We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis.

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.