Formation of Benign Tumours by Stem Cell Misregulation

Our tissues usually have just the right number of cells to optimally fulfil their function. Not enough cells within a tissue can lead to dysfunction, while too many cells result in a tumour. Yet, how this homeostatic balance is maintained remains poorly defined. Most differentiated cells within tissues have a finite lifespan and need to be replaced at a corresponding pace to maintain tissue homeostasis. These new differentiated cells are generated by proliferation of the stem/progenitor cells that serve the tissue. Work in simple invertebrates clearly suggests stem cells respond to at least two types of signals: niche signaling and growth factors. Niche signals promote the undifferentiated state by preventing differentiation, and thus allow for stem cell self-renewal. Growth factor sources comprise a systemic input reflecting the animal’s nutritional status, and a localized, homeostatic feedback from the tissue that the stem cells serve. That homeostatic signal couples stem cell proliferation rates to the tissue’s need for new differentiated cells. Evidence from simple organisms suggests two types of benign tumours can arise from deregulation of either niche or homeostatic signaling. Namely, constitutive niche signaling promotes the formation of undifferentiated “stem cell” tumours, while defective homeostatic signaling leads to the formation of differentiated tumours. We propose that these principles may be conserved and underlie benign tumour formation in humans, while benign tumours can evolve into cancer.

The Proliferation of Pre-Pubertal Porcine Spermatogonia in Stirred Suspension Bioreactors Is Partially Mediated by the Wnt/β-Catenin Pathway

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ β-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling.

Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila

During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

dRTEL1 is essential for the maintenance of Drosophila male germline stem cells

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.

Pharmacological Approaches and Regeneration of Bone Defects with Dental Pulp Stem Cells

Bone defects in the craniomaxillofacial skeleton vary from small periodontal defects to extensive bone loss, which are difficult to restore and can lead to extensive damage of the surrounding structures, deformities, and limited functions. Plenty of surgical regenerative procedures have been developed to reconstruct or prevent alveolar defects, based on guided bone regeneration involving the use of autogenous bone grafts or bone substituents. However, these techniques have limitations in the restoration of morphological and functional reconstruction, thus stopping disease progression but not regenerating lost tissue. Most promising candidates for regenerative therapy of maxillofacial bone defects represent postnatal stem cells, because of their replication potential in the undifferentiated state and their ability to differentiate as well. There is an increased need for using various orofacial sources of stem cells with comparable properties to mesenchymal stem cells because they are more easily available with minimally invasive procedures. In addition to the source of MSCs, another aspect affects the regeneration outcomes. Thermal, mechanical, and chemical stimuli after surgical procedures have the ability to generate pain, usually managed with pharmacological agents, mostly nonsteroidal anti-inflammatory drugs (NSAIDs). Some studies revealed that NSAIDs have no significant cytotoxic effect on bone marrow stem cells from mice, while other studies showed regulation of osteogenic and chondrogenic marker genes in MSC cells by NSAIDs and paracetamol, but no effect was observed in connection with diclofenac use. Therefore, there is a need to focus on such pharmacotherapy, capable of affecting the characteristics and properties of implanted MSCs.

Dermatan-4-O-Sulfotransferase-1 Contributes to the Undifferentiated State of Mouse Embryonic Stem Cells

Mouse embryonic stem cells (mESCs) have the properties of self-renewal and pluripotency. Various signals and growth factors maintain their undifferentiated state and also regulate their differentiation. Glycosaminoglycans are present on the cell surface and in the cell matrix as proteoglycans. Previously, we and other groups reported that the glycosaminoglycan heparan sulfate contributes to both maintenance of undifferentiated state and regulation of mESC differentiation. It has been shown that chondroitin sulfate is needed for pluripotency and differentiation of mESCs, while keratan sulfate is a known marker of human ESCs or induced pluripotent stem cells. We also found that DS promotes neuronal differentiation from mESCs and human neural stem cells; however, the function of DS in the maintenance of mESCs has not yet been revealed. Here, we investigated the role of DS in mESCs by knockdown (KD) or overexpression (O/E) of the dermatan-4-O-sulfotransferase-1 (D4ST1) gene. We found that the activity of the ESC self-renewal marker alkaline phosphatase was reduced in D4ST1 KD mESCs, but, in contrast, increased in D4ST1 O/E mESCs. D4ST1 KD promoted endodermal differentiation, as indicated by an increase in Cdx2 expression. Conversely, Cdx2 expression was decreased by D4ST1 O/E. Wnt signaling, which is also involved in endodermal differentiation, was activated by D4ST1 KD and suppressed by D4ST1 O/E. Collectively, these results demonstrate that D4ST1 contributes to the undifferentiated state of mESCs. Our findings provide new insights into the function of DS in mESCs.

Repurposing melanoma chemotherapy to activate inflammasomes in treatment of BRAF/MAPK inhibitor resistant melanoma

The development of resistance to treatments of melanoma is commonly associated with upregulation of the MAPK pathway and development of an undifferentiated state. Prior studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by activation of inflammasomes. In the present studies we have taken cell lines from patients before and after development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested in vitro and in vivo against xenografts in NSG mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2, NLRP3 or NLRC4 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome targeted therapy.

An N-Cadherin 2 expressing epithelial cell subpopulation predicts response to surgery, chemotherapy and immunotherapy in bladder cancer

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.

Differentiated SH-SY5Y Cells for High-Throughput Screening in a 96-Well Plate Format: Protocol Optimisation and Application for Neurite Outgrowth Assays

Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe a protocol to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 hour improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of this protocol by optimising it further for neurite outgrowth analysis. The presented protocol achieves homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.