Editorial: Tumor microenvironment in primary brain cancers

Primary brain tumors are most commonly detected in the late stages of the disease and there are only a few treatment opportunities. Overall survival rates are significantly low and the response to chemo/radiotherapy is not sufficient, despite surgical intervention. The tumor microenvironment (TME) regulates several brain tumor hallmarks, such as cell migration, invasiveness, proliferation, therapy resistance, stemness maintenance, immune evasion, among others. Therefore, it is necessary to understand the pro-tumoral mechanisms that are regulated by the TME in order to detect and identify new biomarkers and novel therapeutic targets.IN was supported by ANID/FONDECYT Grant 11220149 and ANID/IMII ICN09-016/ICN 2021-045. JRP was supported by grant PID2019-104766RB-C21 funded by MCIN/AEI/10.13039/501100011033, Basque Government (IT1751-22) and University of the Basque Country (COLAB22/07)

Stem and Cancer Stem Cell Identities, Cellular Markers, Niche Environment and Response to Treatments to Unravel New Therapeutic Targets

Adult stem cells are a partially quiescent cell population responsible for natural cell renewal and are found in many different regions of the body, including the brain, teeth, bones, muscles, skin, and diverse epithelia, such as the epidermal or intestinal epithelium, among others [...

shinyCurves, a shiny web application to analyse multisource qPCR amplification data: a COVID‑19 case study

[EN]Background Quantitative, reverse transcription PCR (qRT-PCR) is currently the gold-standard for SARS-CoV-2 detection and it is also used for detection of other virus. Manual data analysis of a small number of qRT-PCR plates per day is a relatively simple task, but automated, integrative strategies are needed if a laboratory is dealing with hundreds of plates per day, as is being the case in the COVID-19 pandemic. Results Here we present shinyCurves, an online shiny-based, free software to analyze qRT-PCR amplification data from multi-plate and multi-platform formats. Our shiny application does not require any programming experience and is able to call samples Positive, Negative or Undetermined for viral infection according to a number of user-defined settings, apart from providing a complete set of melting and amplification curve plots for the visual inspection of results. Conclusions shinyCurves is a flexible, integrative and user-friendly software that speeds-up the analysis of massive qRT-PCR data from different sources, with the possibility of automatically producing and evaluating melting and amplification curve plots.This project was supported by funding from the UPV/EHU (Accion Especial "Desarrollo e implementacion del test de diagnostico para COVID-19"). The funding body did not play any roles in the study design; nor in the data collection, analysis and interpretation, or in the writing of the paper

Vasculogenesis from Human Dental Pulp Stem Cells Grown in Matrigel with Fully Defined Serum-Free Culture Media

The generation of vasculature is one of the most important challenges in tissue engineering and regeneration. Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types to induce vasculogenesis and angiogenesis as they not only secrete vascular endothelial growth factor (VEGF) but can also differentiate in vitro into both endotheliocytes and pericytes in serum-free culture media. Moreover, hDPSCs can generate complete blood vessels containing both endothelial and mural layers in vivo, upon transplantation into the adult brain. However, many of the serum free media employed for the growth of hDPSCs contain supplements of an undisclosed composition. This generates uncertainty as to which of its precise components are necessary and which are dispensable for the vascular differentiation of hDPSCs, and also hinders the transfer of basic research findings to clinical cell therapy. In this work, we designed and tested new endothelial differentiation media with a fully defined composition using standard basal culture media supplemented with a mixture of B27, heparin and growth factors, including VEGF-A165 at different concentrations. We also optimized an in vitro Matrigel assay to characterize both the ability of hDPSCs to differentiate to vascular cells and their capacity to generate vascular tubules in 3D cultures. The description of a fully defined serum-free culture medium for the induction of vasculogenesis using human adult stem cells highlights its potential as a relevant innovation for tissue engineering applications. In conclusion, we achieved efficient vasculogenesis starting from hDPSCs using serum-free culture media with a fully defined composition, which is applicable for human cell therapy purposes.This work was financed by the «Ramón y Cajal» program RYC-2013-13450 (JRP); UPV/EHU (GIU16/66, UFI 11/44 and COLAB19/03; FU); MINECO Retos I + D + I (SAF2015-70866-R; JRP, PID2019-104766RB-C21; JRP); Basque Government (GV/EJ; IT831-13; GI) TERSAFURNA-2020333039 and ELKARTEK KK-2019-00093. J.L. has a grant from UPV/EHU (DOKBERRI 2019(DOCREC19/49)). I.I. has a grant from the Basque Government (PRE_2019_2_0300 or GIU16/66)

Human DenPulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain

Dental pulp stem cells (DPSCs) have the capacity to give rise to cells with neuronal-like phenotypes, suggesting their use in brain cell therapies. In the present work, we wanted to address the phenotypic fate of adult genetically unmodified human DPSCs cultured in Neurocult (TM) (Stem Cell Technologies), a cell culture medium without serum which can be alternatively supplemented for the expansion and/or differentiation of adult neural stem cells (NSCs). Our results show that non-genetically modified human adult DPSCs cultured with Neurocult NS-A proliferation supplement generated neurosphere-like dentospheres expressing the NSC markers Nestin and glial fibrillary acidic protein (GFAP), but also the vascular endothelial cell marker CD31. Remarkably, 1 month after intracranial graft into athymic nude mice, human CD31+/CD146+ and Nestin+ DPSC-derived cells were found tightly associated with both the endothelial and pericyte layers of brain vasculature, forming full blood vessels of human origin which showed an increased laminin staining. These results are the first demonstration that DPSC-derived cells contributed to the generation of neovasculature within brain tissue, and that Neurocult and other related serum-free cell culture media may constitute a fast and efficient way to obtain endothelial cells from human DPSCs.This work was funded by "Ramon y Cajal" program RYC-2013-13450 (JRP) and RYC 2012-11137 (JME); Spanish Ministry of Economy and Competitiveness SAF2015-70866-R; UPV/EHU (GIU16/66, UFI 11/44); and Basque Government (GV/EJ; IT831-13). JL and OP-A obtained a Ph.D. fellowship from the University of the Basque Country (UPV/EHU)

Advances and Perspectives in Dental Pulp Stem Cell Based Neuroregeneration Therapies

Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types for regenerative therapies given their ability to grow in the absence of serum and their realistic possibility to be used in autologous grafts. In this review, we describe the particular advantages of hDPSCs for neuroregenerative cell therapies. We thoroughly discuss the knowledge about their embryonic origin and characteristics of their postnatal niche, as well as the current status of cell culture protocols to maximize their multilineage differentiation potential, highlighting some common issues when assessing neuronal differentiation fates of hDPSCs. We also review the recent progress on neuroprotective and immunomodulatory capacity of hDPSCs and their secreted extracellular vesicles, as well as their combination with scaffold materials to improve their functional integration on the injured central nervous system (CNS) and peripheral nervous system (PNS). Finally, we offer some perspectives on the current and possible future applications of hDPSCs in neuroregenerative cell therapies.This research was supported by MICINN retos I+D+i (PID2019-104766RB-C21 and RYC-2013-13450, to J.R.P.) and UPV/EHU (GIU16/66 and PPGA20/22, to F.U., G.I.; and COLAB19/03 and IKERTU-2020.0155, to F.U., J.R.S.). Y.P. was funded by a Bikaintek PhD grant from the Basque Government (20-AF-W2-2018-00001)

A Comparative Study of Cell Culture Conditions during Conversion from Primed to Naive Human Pluripotent Stem Cells

The successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) represented a turning point in the stem cell research field, owing to their ability to differentiate into any cell type with fewer ethical issues than human embryonic stem cells (hESCs). In mice, PSCs are thought to exist in a naive state, the cell culture equivalent of the immature pre-implantation embryo, whereas in humans, PSCs are in a primed state, which is a more committed pluripotent state than a naive state. Recent studies have focused on capturing a similar cell stage in human cells. Given their earlier developmental stage and therefore lack of cell-of-origin epigenetic memory, these cells would be better candidates for further re-differentiation, use in disease modeling, regenerative medicine and drug discovery. In this study, we used primed hiPSCs and hESCs to evaluate the successful establishment and maintenance of a naive cell stage using three different naive-conversion media, both in the feeder and feeder-free cells conditions. In addition, we compared the directed differentiation capacity of primed and naive cells into the three germ layers and characterized these different cell stages with commonly used pluripotent and lineage-specific markers. Our results show that, in general, naive culture NHSM medium (in both feeder and feeder-free systems) confers greater hiPSCs and hESCs viability and the highest naive pluripotency markers expression. This medium also allows better cell differentiation cells toward endoderm and mesoderm.This work was supported by the Health Department of the Basque Government (Grant 2019111068, 2019/4703, 2020111058, 2020333032, 2021333057 and 2021333012), Merck-Salud Founda- tion (FSALUD17/004), Economic Development and Infrastructures Department of the Basque Govern- ment (KK-2020/00068), EITB Maratoia (BIO21/COV/030), Project “PI18/01299” and “PI21/01187”, funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF) “A way to make Europe”, “ICI21/00095” funded by Instituto de Salud Carlos III and co-funded by European Union (NextGenerationEU), “Plan de Recuperación Transformación y Resiliencia” Investigación Clínica Independiente 2021–Acción Estratégica Salud 2017–2020, RICORS: (RD21/00017/0024) Red Española de Terapias Avanzadas TERAV ISCIII. Funded by Instituto de Salud Carlos III (ISCIII) and co-funded by European Union (NextGenerationEU) “Plan de Recuperación Transformación y Resiliencia” Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) 2021–Acción Estratégica Salud 2017–2020. L.H. was supported by the Jesus Gangoiti Barrera Foundation and the Asociación Española contra el Cáncer (AECC) AECC16/501 and the Fundación Mutua Madrileña AP176182020. M.M-I was supported by Jesus Gangoiti Barrera Foundation. I.R was supported by Margarita Salas Grant “MARSA21/60” and the Jesus Gangoiti Barrera Foundation. M.I-F. was supported by Inocente Inocente Foundation FII18/003. J.R.P. has grant “RYC-2013-13450” funded by MCIN/AEI/10.13039/501100011033, by the European Social Fund “ESF investing in your future”

Is There Such a Thing as a Genuine Cancer Stem Cell Marker? Perspectives from the Gut, the Brain and the Dental Pulp

The conversion of healthy stem cells into cancer stem cells (CSCs) is believed to underlie tumor relapse after surgical removal and fuel tumor growth and invasiveness. CSCs often arise from the malignant transformation of resident multipotent stem cells, which are present in most human tissues. Some organs, such as the gut and the brain, can give rise to very aggressive types of cancers, contrary to the dental pulp, which is a tissue with a very remarkable resistance to oncogenesis. In this review, we focus on the similarities and differences between gut, brain and dental pulp stem cells and their related CSCs, placing a particular emphasis on both their shared and distinctive cell markers, including the expression of pluripotency core factors. We discuss some of their similarities and differences with regard to oncogenic signaling, telomerase activity and their intrinsic propensity to degenerate to CSCs. We also explore the characteristics of the events and mutations leading to malignant transformation in each case. Importantly, healthy dental pulp stem cells (DPSCs) share a great deal of features with many of the so far reported CSC phenotypes found in malignant neoplasms. However, there exist literally no reports about the contribution of DPSCs to malignant tumors. This raises the question about the particularities of the dental pulp and what specific barriers to malignancy might be present in the case of this tissue. These notable differences warrant further research to decipher the singular properties of DPSCs that make them resistant to transformation, and to unravel new therapeutic targets to treat deadly tumors.This work has been financed by The University of The Basque Country (UPV/EHU): Grant number GIU16/66, UFI 11/44, COLAB19/03 and IKERTU-2020.0155 (to F.U), the Basque Government/Eusko Jaurkaritza: ELKARTEK KK-2019/00093 (to U.F.), and MINECO “Ramón y Cajal” program RYC-2013-13450 and MINECO PID2019-104766RB-C21 (to P.J.R.). L.J. was funded by a UPV/EHU postdoctoral fellowship DOKBERRI 2019 (DOCREC19/49) program

Enhanced Adipogenic Differentiation of Human Dental Pulp Stem Cells in Enzymatically Decellularized Adipose Tissue Solid Foams

Engineered 3D human adipose tissue models and the development of physiological human 3D in vitro models to test new therapeutic compounds and advance in the study of pathophysiological mechanisms of disease is still technically challenging and expensive. To reduce costs and develop new technologies to study human adipogenesis and stem cell differentiation in a controlled in vitro system, here we report the design, characterization, and validation of extracellular matrix (ECM)-based materials of decellularized human adipose tissue (hDAT) or bovine collagen-I (bCOL-I) for 3D adipogenic stem cell culture. We aimed at recapitulating the dynamics, composition, and structure of the native ECM to optimize the adipogenic differentiation of human mesenchymal stem cells. hDAT was obtained by a two-enzymatic step decellularization protocol and post-processed by freeze-drying to produce 3D solid foams. These solid foams were employed either as pure hDAT, or combined with bCOL-I in a 3:1 proportion, to recreate a microenvironment compatible with stem cell survival and differentiation. We sought to investigate the effect of the adipogenic inductive extracellular 3D-microenvironment on human multipotent dental pulp stem cells (hDPSCs). We found that solid foams supported hDPSC viability and proliferation. Incubation of hDPSCs with adipogenic medium in hDAT-based solid foams increased the expression of mature adipocyte LPL and c/EBP gene markers as determined by RT-qPCR, with respect to bCOL-I solid foams. Moreover, hDPSC capability to differentiate towards adipocytes was assessed by PPAR-γ immunostaining and Oil-red lipid droplet staining. We found out that both hDAT and mixed 3:1 hDAT-COL-I solid foams could support adipogenesis in 3D-hDPSC stem cell cultures significantly more efficiently than solid foams of bCOL-I, opening the possibility to obtain hDAT-based solid foams with customized properties. The combination of human-derived ECM biomaterials with synthetic proteins can, thus, be envisaged to reduce fabrication costs, thus facilitating the widespread use of autologous stem cells and biomaterials for personalized medicine.This research was funded by the Basque Government (IT1751-22; to G.I.; ELKARTEK program 566 PLAKA KK-2019-00093; to N.B.), the Health Department of the Basque Government (grant No. 2021333012; to J.R.P.), and grant No. RYC-2013-13450 and grant No. PID2019-104766RB-C21 funded by MCIN/AEI/10.13039/501100011033 by the European Union (NextGenerationEU) “Plan de Recuperación Transformación y Resiliencia” (grants to J.R.P.)

Osteogenic differentiation of human dental pulp stem cells in decellularised adipose tissue solid foams

3D cell culture systems based on biological scaffold materials obtainable from both animal and human tissues constitute very interesting tools for cell therapy and personalised medicine applications. The white adipose tissue (AT) extracellular matrix (ECM) is a very promising biomaterial for tissue engineering due to its easy accessibility, malleability and proven biological activity. In the present study, human dental pulp stem cells (hDPSCs) were combined in vitro with ECM scaffolds from porcine and human decellularised adipose tissues (pDAT, hDAT) processed as 3D solid foams, to investigate their effects on the osteogenic differentiation capacity and bone matrix production of hDPSCs, compared to single-protein-based 3D solid foams of collagen type I and conventional 2D tissue-culture-treated polystyrene plates. pDAT solid foams supported the osteogenic differentiation of hDPSCs to similar levels to collagen type I, as assessed by alkaline phosphatase and alizarin red stainings, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and osteocalcin/bone gamma-carboxyglutamate protein (BGLAP) immunostaining. Interestingly, hDAT solid foams showed a markedly lower capacity to sustain hDPSC osteogenic differentiation and matrix calcification and a higher capacity to support adipogenesis, as assessed by RT-qPCR and oil red O staining. White ATs from both human and porcine origins are relatively abundant and available sources of raw material to obtain high quality ECM-derived biomedical products. These biomaterials could have promising applications in tissue engineering and personalised clinical therapy for the healing and regeneration of lesions involving not only a loss of calcified bone but also its associated soft non-calcified tissues.This research was supported by the Basque Government (ELKARTEK program PLAKA KK- 2019-00093; to NB), MICINN retos I+D+i (PID2019- 104766RB-C21, to JRP) and UPV/EHU (PPGA20/22; to FU, GI). The authors would like to thank the staff members of the SGIKER services of the UPV/EHU: Lipidomic service (Beatriz Abad) and Analytical Microscopy (Ricardo Andrade, Alejandro Díez-Torre and Irene Fernández) for their technical assistance