Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development.

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

Spontaneous Osteoclastogenesis, a risk factor for bone metastasis in advanced luminal A-type breast cancer patients.

Introduction: Osteolytic bone metastasis in advanced breast cancer stages are a major complication for patient´s quality life and a sign of low survival prognosis. Permissive microenvironments which allow cancer cell secondary homing and later proliferation are fundamental for metastatic processes. The causes and mechanisms behind bone metastasis in breast cancer patients are still an unsolved puzzle. Therefore, in this work we contribute to describe bone marrow pre-metastatic niche in advanced breast cancer patients. Results: We show an increase in osteoclasts precursors with a concomitant imbalance towards spontaneous osteoclastogenesis which can be evidenced at bone marrow and peripheral levels. Pro-osteoclastogenic factors RANKL and CCL-2 may contribute to bone resorption signature observed in bone marrow. Meanwhile, expression levels of specific microRNAs in primary breast tumors may already indicate a pro-osteoclastogenic scenario prior to bone metastasis. Discussion: The discovery of prognostic biomarkers and novel therapeutic targets linked to bone metastasis initiation and development are a promising perspective for preventive treatments and metastasis management in advanced breast cancer patients.

A novel approach for studying mast cell-driven disorders: Mast cells derived from induced pluripotent stem cells.

BACKGROUND: Mast cells (MCs) are considered the main effectors in allergic reactions and well known for their contribution to the pathogenesis of various inflammatory diseases, urticaria, and mastocytosis. To study their functions in vitro, human primary MCs are isolated directly from several tissues or differentiated from hematopoietic progenitors. However, these techniques bear several disadvantages and challenges including low proliferation capacity, donor-dependent heterogeneity, and the lack of a continuous cell source. OBJECTIVE: To address this, we developed a novel strategy for the rapid and efficient differentiation of MCs from human-induced pluripotent stem cells (hiPSCs). METHODS: A 4-step protocol for the generation of hiPSC-derived MCs, based on the use of 3 hiPSC lines, was established and validated by comparison with human skin MCs and peripheral hematopoietic stem cell-derived MCs. RESULTS: hiPSC-MCs share phenotypic and functional characteristics of human skin MCs and peripheral hematopoietic stem cell-derived MCs. They display stable expression of the MC-associated receptors CD117, FcεRIα, and Mas-related G protein-coupled receptor X2 and degranulate in response to IgE/anti-IgE and substance P. CONCLUSIONS: This novel hiPSC-based approach provides a sustainable and homogeneous source for a rapid and highly productive generation of phenotypically mature, functional MCs, and its principle allows for the investigation of disease- and patient-specific MC populations.

Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC) -derived sensory neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent, potentially irreversible adverse effect of cytotoxic chemotherapy often leading to a reduction or discontinuation of treatment which negatively impacts patients' prognosis. To date, however, neither predictive biomarkers nor preventive treatments for CIPN are available, which is partially due to a lack of suitable experimental models. We therefore aimed to evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for CIPN. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to axonal blebbing and a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not observed for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. Paclitaxel treatment effects were less pronounced after 24 h but prominent when treatment was applied for 72 h. Global transcriptome analyses performed at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways. We further evaluated if known neuroprotective strategies can be reproduced in iPSC-DSN and observed protective effects of lithium replicating findings from rodent dorsal root ganglia cells. Comparing sensory neurons derived from two different healthy donors, we found preliminary evidence that these cell lines react differentially to neurotoxic drugs as expected from the variable presentation of CIPN in patients. In conclusion, iPSC-DSN are a promising platform to study the pathogenesis of CIPN and to evaluate neuroprotective treatment strategies. In the future, the application of patient-specific iPSC-DSN could open new avenues for personalized medicine with individual risk prediction, choice of chemotherapeutic compounds and preventive treatments.

Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2(+) cells generating gastric spheroids differed from adult glandular Lgr5(+) stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5(+) stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2(+) cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2(+) cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

CD105 expression on CD34-negative spindle-shaped stromal cells of primary tumor is an unfavorable prognostic marker in early breast cancer patients.

Several studies have confirmed that the breast tumor microenvironment drives cancer progression and metastatic development. The aim of our research was to investigate the prognostic significance of the breast tumor microenvironment in untreated early breast cancer patients. Therefore, we analyzed the association of the expression of α-SMA, FSP, CD105 and CD146 in CD34-negative spindle-shaped stromal cells, not associated with the vasculature, in primary breast tumors with classical prognostic marker levels, metastatic recurrence, local relapse, disease-free survival, metastasis-free survival and the overall survival of patients. In the same way, we evaluated the association of the amount of intra-tumor stroma, fibroblasts, collagen deposition, lymphocytic infiltration and myxoid changes in these samples with the clinical-pathological data previously described. This study is the first to demonstrate the high CD105 expression in this stromal cell type as a possible independent marker of unfavorable prognosis in early breast cancer patients. Our study suggests that this new finding can be useful prognostic marker in the clinical-pathological routine.

Association between ligands and receptors related to the progression of early breast cancer in tumor epithelial and stromal cells.

BACKGROUND: Despite advances in the study of breast cancer (BC), it remains the second leading cause of mortality among women. BC is a heterogeneous system, mainly composed of tumor epithelial cells (TEpCs) and stromal cells (SCs); the interaction through the ligands and their receptors (Rs) plays a major role in BC progression. The aim of the present study was to evaluate the association between ligands, such as osteoprotegerin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand (RANKL), stromal cell-derived factor (SDF)-1, interleukin (IL)-6, macrophage colony stimulating factor, chemokine (C-C motif) ligand-2 (CCL-2), and their Rs in TEpC and spindle-shaped SCs not closely associated with the vasculature. PATIENTS AND METHODS: We studied the expression of all those factors in 63 primary tumors of untreated patients with BC with infiltrative ductal carcinoma (I/II stage) and 10 non-neoplastic tissues. The percentage of positive cells and the staining intensity were analyzed by immunohistochemistry. Mann-Whitney test and Spearman's rank correlation coefficient were used (P ≤ .05). RESULTS: We found a significant association between the expression of RANKL, IL-6, SDF-1, and CCL-2 in TEpC and the receptor activator of nuclear factor kappa B (RANK), IL-6R, C-X-C chemokine R type 4, and chemokine (C-C motif) R-2 (CCR-2) in spindle-shaped SC. The expression of TRAIL, RANKL, and CCL-2 in spindle-shaped SC also was associated with the expression of TRAIL-receptor 1, TRAIL-receptor 4, RANK, and CCR-2 in TEpC. CONCLUSIONS: Because the described ligands and Rs are implicated in BC progression, our results suggest that these factors could be involved in the crosstalk between TEpC and SC in the early stages of BC.

Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.

Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

Behaviour of mesenchymal stem cells from bone marrow of untreated advanced breast and lung cancer patients without bone osteolytic metastasis.

Tumour cells can find in bone marrow (BM) a niche rich in growth factors and cytokines that promote their self-renewal, proliferation and survival. In turn, tumour cells affect the homeostasis of the BM and bone, as well as the balance among haematopoiesis, osteogenesis, osteoclastogenesis and bone-resorption. As a result, growth and survival factors normally sequestered in the bone matrix are released, favouring tumour development. Mesenchymal stem cells (MSCs) from BM can become tumour-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastasis. In this study we showed that BM MSCs from untreated advanced breast and lung cancer patients, without bone metastasis, had low osteogenic and adipogenic differentiation capacity compared to that of healthy volunteers. In contrast, chondrogenic differentiation was increased. Moreover, MSCs from patients had lower expression of CD146. Finally, our data showed higher levels of Dkk-1 in peripheral blood plasma from patients compared with healthy volunteers. Because no patient had any bone disorder by the time of the study we propose that the primary tumour altered the plasticity of MSCs. As over 70 % of advanced breast cancer patients and 30-40 % of lung cancer patients will develop osteolytic bone metastasis for which there is no total cure, our findings could possibly be used as predictive tools indicating the first signs of future bone disease. In addition, as the MSCs present in the BM of these patients may not be able to regenerate bone after the tumour cells invasion into BM/bone, it is possible that they promote the cycle between tumour cell growth and bone destruction.