Insights from multi-omics integration in complex disease primary tissues.

Genome-wide association studies (GWAS) have provided insights into the genetic basis of complex diseases. In the next step, integrative multi-omics approaches can characterize molecular profiles in relevant primary tissues to reveal the mechanisms that underlie disease development. Here, we highlight recent progress in four examples of complex diseases generated by integrative studies: type 2 diabetes (T2D), osteoarthritis, Alzheimer's disease (AD), and systemic lupus erythematosus (SLE). High-resolution methodologies such as single-cell and spatial omics techniques will become even more important in the future. Furthermore, we emphasize the urgent need to include as yet understudied cell types and increase the diversity of studied populations.

Human Primary Airway Basal Cells Display a Continuum of Molecular Phases from Health to Disease in Chronic Obstructive Pulmonary Disease.

Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.

Regenerative responses of rabbit corneal endothelial cells to stimulation by fibroblast growth factor 1 (FGF1) derivatives, TTHX1001 and TTHX1114.

Utilising rabbit corneal endothelial cells (CEC) in three different paradigms, two human FGF1 derivatives (TTHX1001 and TTHX1114), engineered to exhibit greater stability, were tested as proliferative agents. Primary CECs and mouse NIH 3T3 cells treated with the two FGF1 derivatives showed equivalent EC50 ranges (3.3-24 vs.1.9-16. ng/mL) and, in organ culture, chemically lesioned corneas regained half of the lost endothelial layer in three days after treatment with the FGF1 derivatives as compared to controls. In vivo, following cryolesioning, the CEC monolayer, as judged by specular microscopy, regenerated 10-11 days faster when treated with TTHX1001. Over two weeks, all treated eyes showed clearing of opacity about twice that of untreated controls. In all three rabbit models, both FGF1 derivatives were effective in inducing CEC proliferation over control conditions, supporting the prediction that these stabilised FGF1 derivatives can potentially regenerate corneal endothelial deficits in humans.

High fidelity hypothermic preservation of primary tissues in organ transplant preservative for single cell transcriptome analysis.

BACKGROUND: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards. RESULTS: Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution. CONCLUSIONS: Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.

Ex vivo culture of primary human colonic tissue for studying transcriptional responses to 1α,25(OH)2 and 25(OH) vitamin D.

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is a steroid hormone derived from circulating 25(OH) vitamin D [25(OH)D] with chemopreventive effects in colorectal cancer. 1α,25(OH)2D3 acts through transcriptional mechanisms; however, our understanding of vitamin D transcriptional responses in the colon is derived from studies in transformed cancer cell lines which may not represent responses in normal healthy tissue. Here, we describe the optimization of an ex vivo culture model using primary colonic biopsy samples for studying short-term transcriptional response induced by 1α,25(OH)2D3 and 25(OH)D treatment. Colon biopsy samples from healthy subjects were maintained in primary culture and treated in parallel with 100 nM 1α,25(OH)2D3 or 62.5 nM 25(OH)D and vehicle control (ethanol). Viability was assessed using histology and enzymatic assays. Genome-wide transcriptional responses to 1α,25(OH)2D3 were assessed and expression of 25(OH)D targets CYP27B1 and CYP24A1 were measured by real time PCR. We show that ex vivo culture of colonic tissue remains viable for up to 8 h. The largest number of differentially expressed genes in response to 1α,25(OH)2D3 was noted after 6 h (n = 120). As proof of concept, the top upregulated gene was CYP24A1, a well-established vitamin D-responsive gene. With 25(OH)D treatment, mRNA expression of CYP27B1 was significantly increased after 1 h, while expression of CYP24A1 was greatest at 8 h. Ex vivo culture can be used to assess short-term transcriptional responses to 1α,25(OH)2D3 and 25(OH)D in primary tissue from human colon. Future studies will address interindividual differences in transcriptional responses.

Fibroblast Isolation from Mammary Gland Tissue and Syngeneic Murine Breast Cancer Models.

Cancer-associated fibroblasts (CAFs) are vital within the tumor ecosystem, regulating tumor growth, dissemination, and response to therapy through crosstalk with tumor cells, infiltrating immune and vascular cells, as well as components of the extracellular matrix (ECM). CAFs have thus emerged as potential therapeutic targets to complement cancer cell-targeted therapies. To study CAF-tumor cell crosstalk ex vivo, robust isolation methods of primary CAFs are required. Here, we present protocols to isolate, expand, and culture two types of fibroblasts: (1) healthy murine mammary gland fibroblasts, a key source of the CAF population in breast tumor models and (2) CAFs derived from syngeneic murine breast tumors. Isolated mammary fibroblasts and CAFs are suitable for use in a variety of downstream cellular and molecular experiments. We expect these methods to be useful to scientists studying the properties of fibroblasts and CAFs and the interaction between CAFs and the various components of the tumor microenvironment (TME).

Primary human organoids models: Current progress and key milestones.

During the past 10 years the world has experienced enormous progress in the organoids field. Human organoids have shown huge potential to study organ development, homeostasis and to model diseases in vitro. The organoid technology has been widely and increasingly applied to generate patient-specific in vitro 3D cultures, starting from both primary and reprogrammed stem/progenitor cells. This has consequently fostered the development of innovative disease models and new regenerative therapies. Human primary, or adult stem/progenitor cell-derived, organoids can be derived from both healthy and pathological primary tissue samples spanning from fetal to adult age. The resulting 3D culture can be maintained for several months and even years, while retaining and resembling its original tissue's properties. As the potential of this technology expands, new approaches are emerging to further improve organoid applications in biology and medicine. This review discusses the main organs and tissues which, as of today, have been modelled in vitro using primary organoid culture systems. Moreover, we also discuss the advantages, limitations, and future perspectives of primary human organoids in the fields of developmental biology, disease modelling, drug testing and regenerative medicine.

Looking for biomarkers in interferon response pathway to predict response to oncolytic HSV-1 in breast cancer: An ex vivo study.

Breast cancer is the most common malignancy in women worldwide. Administration of oncolytic viruses is one of the novel promising cancer therapy approaches. Replication of these viruses is usually limited to cancer cells that have interferon (IFN) signaling defects. However, Interferon signaling is not completely impaired in all cancer cells which may limit the benefits of virotherapy.    Identification of realistic IFN-mediated biomarkers to identify patients who most likely respond to virotherapy would be helpful. In this study, eight patients-derived primary tumor cultures were infected with an ICP34.5 deleted oHSV, then the rate of infectivity, cell survival, and expression of the gene involved in IFN pathway were analyzed.Data showed that mRNA expressions of Myeloid differentiation primary response protein (Myd88) is significantly higher in tumors whose primary cultures showed less cell death and resistance to oHSV infectivity (P-value < 0.05). The differentiating cut off of Myd88 expression, inferred from the receiver operating characteristic (ROC) curve, predicted that only 13 out of 16 other patients could be sensitive to this oHSV. Identifying such biomarker improves our ability to select the patients who do not exhibit resistance to virotherapy.

A Potential New Treatment for High-Grade Glioma: A Study Assessing Repurposed Drug Combinations against Patient-Derived High-Grade Glioma Cells.

Repurposed drugs have demonstrated in vitro success against high-grade gliomas; however, their clinical success has been limited due to the in vitro model not truly representing the clinical scenario. In this study, we used two distinct patient-derived tumour fragments (tumour core (TC) and tumour margin (TM)) to generate a heterogeneous, clinically relevant in vitro model to assess if a combination of repurposed drugs (irinotecan, pitavastatin, disulfiram, copper gluconate, captopril, celecoxib, itraconazole and ticlopidine), each targeting a different growth promoting pathway, could successfully treat high-grade gliomas. To ensure the clinical relevance of our data, TC and TM samples from 11 different patients were utilized. Our data demonstrate that, at a concentration of 100µm or lower, all drug combinations achieved lower LogIC50 values than temozolomide, with one of the combinations almost eradicating the cancer by achieving cell viabilities below 4% in five of the TM samples 6 days after treatment. Temozolomide was unable to stop tumour growth over the 14-day assay, while combination 1 stopped tumour growth, with combinations 2, 3 and 4 slowing down tumour growth at higher doses. To validate the cytotoxicity data, we used two distinct assays, end point MTT and real-time IncuCyte life analysis, to evaluate the cytotoxicity of the combinations on the TC fragment from patient 3, with the cell viabilities comparable across both assays. The local administration of combinations of repurposed drugs that target different growth promoting pathways of high-grade gliomas have the potential to be translated into the clinic as a novel treatment strategy for high-grade gliomas.

Enrichment of Cancer Stem Cells in a Tumorsphere Assay.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. CSCs possess tumor initiation potential as well as the ability to resist toxic compounds and chemotherapeutic agents through the upregulation of drug efflux transporters, DNA repair pathways, and survival cascades. Accumulating evidence suggests that CSCs are responsible for tumor relapse and resistance to chemotherapeutic agents and that targeting CSCs is critical to inhibition of cancer progression. Therefore, isolation and characterization of CSCs is important in studying tumor initiation and progression. In this chapter, we provide a detailed method for the identification and isolation of CSCs.