Rationale for combined therapies in severe-to-critical COVID-19 patients.

An unprecedented global social and economic impact as well as a significant number of fatalities have been brought on by the coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acute SARS-CoV-2 infection can, in certain situations, cause immunological abnormalities, leading to an anomalous innate and adaptive immune response. While most patients only experience mild symptoms and recover without the need for mechanical ventilation, a substantial percentage of those who are affected develop severe respiratory illness, which can be fatal. The absence of effective therapies when disease progresses to a very severe condition coupled with the incomplete understanding of COVID-19's pathogenesis triggers the need to develop innovative therapeutic approaches for patients at high risk of mortality. As a result, we investigate the potential contribution of promising combinatorial cell therapy to prevent death in critical patients.

Understanding autophagy role in cancer stem cell development.

Cancer stem cells (CSCs) are a small subpopulation of immature cells located in the tumor mass. These cells are responsible for tumor development, proliferation, resistance and spreading. CSCs are characterized by three unique features: the ability to self-renew, differentiation and tumor formation. CSCs are similar to stem cells, but they differ in the malignant phenotype. CSCs become immortal and survive harsh environmental conditions such as hypoxia, starvation and oxidative stress. However, this harsh tumor microenvironment induces the activation of autophagy, which further increases the CSCs stemness profile, and all these features further increase tumorigenicity and metastasis capacity. Autophagy is induced by the extracellular and cellular microenvironment. Hypoxia is one of the most common factors that highly increases the activity of autophagy in CSCs. Therefore, hypoxia-induced autophagy and CSCs proliferation should be elucidated in order to find a novel cure to defeat cancer cells (CSCs and non-CSCs). The remaining challenges to close the gap between the laboratory bench and the development of therapies, to use autophagy against CSCs in patients, could be addressed by adopting a 3D platform to better-mimic the natural environment in which these cells reside. Ultimately allowing to obtain the blueprints for bioprocess scaling up and to develop the production pipeline for safe and cost-effective autophagy-based novel biologics.

Tackling Ischemic Reperfusion Injury With the Aid of Stem Cells and Tissue Engineering.

Ischemia is a severe condition in which blood supply, including oxygen (O), to organs and tissues is interrupted and reduced. This is usually due to a clog or blockage in the arteries that feed the affected organ. Reinstatement of blood flow is essential to salvage ischemic tissues, restoring O, and nutrient supply. However, reperfusion itself may lead to major adverse consequences. Ischemia-reperfusion injury is often prompted by the local and systemic inflammatory reaction, as well as oxidative stress, and contributes to organ and tissue damage. In addition, the duration and consecutive ischemia-reperfusion cycles are related to the severity of the damage and could lead to chronic wounds. Clinical pathophysiological conditions associated with reperfusion events, including stroke, myocardial infarction, wounds, lung, renal, liver, and intestinal damage or failure, are concomitant in due process with a disability, morbidity, and mortality. Consequently, preventive or palliative therapies for this injury are in demand. Tissue engineering offers a promising toolset to tackle ischemia-reperfusion injuries. It devises tissue-mimetics by using the following: (1) the unique therapeutic features of stem cells, i.e., self-renewal, differentiability, anti-inflammatory, and immunosuppressants effects; (2) growth factors to drive cell growth, and development; (3) functional biomaterials, to provide defined microarchitecture for cell-cell interactions; (4) bioprocess design tools to emulate the macroscopic environment that interacts with tissues. This strategy allows the production of cell therapeutics capable of addressing ischemia-reperfusion injury (IRI). In addition, it allows the development of physiological-tissue-mimetics to study this condition or to assess the effect of drugs. Thus, it provides a sound platform for a better understanding of the reperfusion condition. This review article presents a synopsis and discusses tissue engineering applications available to treat various types of ischemia-reperfusions, ultimately aiming to highlight possible therapies and to bring closer the gap between preclinical and clinical settings.

Adoptive transfer of ex vivo expanded SARS-CoV-2-specific cytotoxic lymphocytes: A viable strategy for COVID-19 immunosuppressed patients?

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is on focus of research. We evaluate herein the feasibility of expanding virus-specific T cells (VST) against SARS-CoV-2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS-CoV-2 asymptomatic infection/negative serology, (b) SARS-CoV-2 symptomatic infection/positive serology, and (c) no history of SARS-CoV-2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T-cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof-of-concept study supports the feasibility of expanding ex vivo SARS-CoV-2-specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients.

Allogeneic hematopoietic stem cell transplant recipients in Spain: Human leukocyte antigen characteristics and diversity by high-resolution analysis.

There are many studies on the polymorphism of the HLA system in healthy donor populations, such as registries of unrelated bone marrow donors. Investigations on the characterization of the HLA complex in hematopoietic stem cell transplant (HSCT) patients, however, are scarce, at least in the Spanish population. This study presents a large-scale analysis of allelic diversity and HLA distribution at a high-resolution level in 2886 patients undergoing HSCT in Spanish centres of the "Grupo Español de Trasplante Hematopoyético y Terapia Celular" during a period of 11 years. Allelic diversity analysis identified 67 HLA-A, 133 HLA-B, 60 HLA-C, 63 HLA-DRB1, 24 HLA-DQB1 and 27 HLA-DPB1 different alleles. Rare alleles were detected among which 33 alleles had not been reported in the European catalog of common and well-documented HLA alleles. Regarding the distribution of five genes-haplotypes, it was observed that the five most frequent extended haplotypes found in our population were between the most common in other Spanish populations, both in patients and in healthy subjects. However, some particular haplotypes were also detected. Bilocus associations HLA-C ~ B and -DRB1 ~ DQB1 were analyzed in order to predict the probability of finding 10/10 matched donors in registries. We found HLA-B alleles showing a great diversity of combinations with HLA-C alleles and unusual associations involving a negative predicting factor. In the field of adoptive therapies, our work supports the necessity to expand further research of TCR-engineered cells, adoptive transfer of virus-specific T-cells and vaccines to target HLA alleles other than A*02:01. HLA alleles such as A*01:01, A*03:01, A*24:02, B*44:03, B*07:02 or B*51:01, might be considered new targets due to its high frequency in our population.

EpCAM duality becomes this molecule in a new Dr. Jekyll and Mr. Hyde tale.

EpCAM, known as an epithelial cell adhesion molecule, plays an essential role in cell adhesion, migration, metastasis and cell signalling. Rather than acting as an apoptosis antagonist, it induces cellular proliferation that impacts the cell cycle, and as a signalling transducer it uses and enhances the Wnt pathway, which is significantly relevant in cell renewal and cancer. EpCAM has become a marker of circulating tumour cells (CTCs) in lung cancer due to its specificity, and its high and stable expression level. Recent findings have allowed us to relearn and discover EpCAM again as a CSCs marker by demonstrating its role in human epithelial cancer progression. In line with this, the focus of attention on EpCAM has become an appealing therapeutic target, although the literature shows a clear controversy in information about its clinical significance. Despite this contradictory fact, solid evidence has demonstrated its dual role as a molecule with oncogenic and tumour suppressor properties, in which the microenvironment is influential. Therefore, its dual role appears to be both tissue- and tumour- dependent. In this review, we summarised the novel and updated insights in the EpCAM field by simplifying the understanding of the biological role of this fascinating molecule, and by showing the promising therapeutic tools that have been developed by various approaches which use antibodies and vaccines for different cancer types for the clear purpose of improving patient outcome.

New insights in non-small-cell lung cancer: circulating tumor cells and cell-free DNA.

Lung cancer is the second most frequent tumor and the leading cause of death by cancer in both men and women. Increasing knowledge about the cancer genome and tumor environment has led to a new setting in which morphological and molecular characterization is needed to treat patients in the most personalized way in order to achieve better outcomes. Since tumor products can be detected in body fluids, the liquid biopsy, particularly, peripheral blood, has emerged as a new source for lung cancer biomarker's analysis. A variety of tumor components can be used for this purpose. Among them, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) should be especially considered. Different detection methods for both CTCs and ctDNA have been and are being developed to improve the sensitivity and specificity of these tests. This would lead to better characterization and would solve some clinical doubts at different disease evolution times, e.g., intratumoral or temporal heterogeneity, difficulty in the obtaining a tumor sample, etc., and would also avoid the side effects of very expensive and complicated tumor obtaining interventions. CTCs and ctDNA are useful in different lung cancer settings. Their value has been shown for the early diagnosis, prognosis, prediction of treatment efficacy, monitoring responses and early detection of lung cancer relapse. CTCs have still not been validated for use in clinical settings in non-small-cell lung cancer (NSCLC), while ctDNA has been approved by the Food and Drug Administration (FDA) and European Medical Association (EMA), and the main clinical guidelines used for detect different epidermal growth factor receptor (EGFR) mutations and the monitoring and treatment choice of mutated patients with tyrosine kinase inhibitors (TKIs). This review, describes how ctDNA seem to be winning the race against CTCs from the laboratory bench to clinical practice due to easier obtaining methods, manipulation and its implementation into clinical practice.

Improving embryonic stem cell expansion through the combination of perfusion and Bioprocess model design.

BACKGROUND: High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement. METHODOLOGY/PRINCIPAL FINDINGS: To characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality 'naïve' mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency ('stemness') genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment. CONCLUSIONS/SIGNIFICANCE: The limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which necessitates the design of suitable ESC bioprocesses. We propose a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.

Cells, stem cells, and cancer stem cells.

The stem cell field owes a great deal to the previous work conducted by embryologists and researchers devoted to reproductive medicine. The time is coming when this emerging field will pay off in the reproductive sciences by offering new avenues of understanding gametogenesis and early embryonic development. Human embryonic stem cells are pluripotent cells that proliferate in vitro while maintaining an undifferentiated state, and they are capable of differentiating into most cell types under appropriate conditions. Embryo-friendly approaches have been developed as new methods of obtaining human embryonic stem cells without destroying the embryo. Somatic stem cells have been identified and isolated from numerous adult organs and tissues to create a multipotent and autologous source of cells with established medical indications. Cell reprogramming is now a scientific fact, and induced pluripotent cells, a new pluripotent cell type, have been generated by the overexpression of specific genes from a myriad of differentiated adult cell types. Cancer is now considered a stem cell disease. Cancer stem cells share numerous features with normal stem cells including hallmarks properties such as self-renewal and undifferentiation. Therefore, the actual focus of ovarian cancer research on the cancer stem cell model should generate efficient and personalized treatment designs to improve treatment efficiency.

Overcoming challenges of ovarian cancer stem cells: novel therapeutic approaches.

Understanding the genetic and molecular mechanisms of ovarian cancer has been the focus of research efforts working toward the greater goal of improving cancer therapy for patients with residual disease after initial treatment with conventional surgery and neoadjuvant chemotherapy. The focus of this review will be centered on new therapeutic strategies based on Cancer Stem Cells studies of chemoresistant subpopulations, the prevention of metastasis, and individualized therapy in order to find the most successful combination of treatments to effectively treat human ovarian cancer. We reviewed recent literature (1993-2011) of novel treatment approaches to ovarian cancer stem cells. As the focus of ovarian cancer investigation has centered on the cancer stem cell model and the complexities that it presents in the development of effective treatments, the future of treating ovarian cancer lies in utilizing individualized treatment systems that include enhancing existing treatments, aiming for novel therapy targets, managing the plasticity of stem cells to induce cellular differentiation, and regulating oncogenic signaling pathways.

Comparison of Cryotip vs. Cryotop for mouse and human blastomere vitrification.

OBJECTIVE: Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos. DESIGN: Experimental prospective controlled study. SETTING: Research center. PATIENT(S): Human Blastomeres were obtained after biopsy. INTERVENTION(S): Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing. MAIN OUTCOME MEASURE(S): Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres. RESULT(S): After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip. CONCLUSION(S): The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives.

Transdifferentiation of MALME-3M and MCF-7 Cells toward Adipocyte-like Cells is Dependent on Clathrin-mediated Endocytosis.

Enforced cell transdifferentiation of human cancer cells is a promising alternative to conventional chemotherapy. We previously identified albumin-associated lipid- and, more specifically, saturated fatty acid-induced transdifferentiation programs in human cancer cells (HCCLs). In this study, we further characterized the adipocyte-like cells, resulting from the transdifferentiation of human cancer cell lines MCF-7 and MALME-3M, and proposed a common mechanistic approach for these transdifferentiating programs. We showed the loss of pigmentation in MALME-3M cells treated with albumin-associated lipids, based on electron microscopic analysis, and the overexpression of perilipin 2 (PLIN2) by western blotting in MALME-3M and MCF-7 cells treated with unsaturated fatty acids. Comparing the gene expression profiles of naive melanoma MALME-3M cells and albumin-associated lipid-treated cells, based on RNA sequencing, we confirmed the transcriptional upregulation of some key adipogenic gene markers and also an alternative splicing of the adipogenic master regulator PPARG, that is probably related to the reported up regulated expression of the protein. Most importantly, these results also showed the upregulation of genes responsible for Clathrin (CLTC) and other adaptor-related proteins. An increase in CLTC expression in the transdifferentiated cells was confirmed by western blotting. Inactivation of CLTC by chlorpromazine (CHP), an inhibitor of CTLC mediated endocytosis (CME), and gene silencing by siRNAs, partially reversed the accumulation of neutral lipids observed in the transdifferentiated cells. These findings give a deeper insight into the phenotypic changes observed in HCCL to adipocyte-like transdifferentiation and point towards CME as a key pathway in distinct transdifferentiation programs. DISCLOSURES: Simon C and Aguilar-Gallardo C are co-inventors of the International Patent Application No. PCT/EP2011/004941 entitled "Methods for tumor treatment and adipogenesis differentiation".

Specific unsaturated fatty acids enforce the transdifferentiation of human cancer cells toward adipocyte-like cells.

Differentiation therapy pursues the discovery of novel molecules to transform cancer progression into less aggressive phenotypes by mechanisms involving enforced cell transdifferentiation. In this study, we examined the identification of transdifferentiating adipogenic programs in human cancer cell lines (HCCLs). Our findings showed that specific unsatturated fatty acids, such as palmitoleic, oleic and linoleic acids, trigger remarkable phenotypic modifications in a large number of human cancer cell lines (HCCLs), including hepatocarcinoma HUH-7, ovarian carcinoma SK-OV-3, breast adenocarcinoma MCF-7 and melanoma MALME-3M. In particular, we characterized a massive biogenesis of lipid droplets (LDs) and up-regulation of the adipogenic master regulator, PPARG, resulting in the transdifferentiation of HCCLs into adipocyte-like cells. These findings suggest the possibility of a novel strategy in cancer differentiation therapy via switching the identity of HCCLs to an adipogenic phenotype through unsaturated fatty acid-induced transdifferentiation.

Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder.

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco's modified Eagle's medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.

Building a framework for embryonic microenvironments and cancer stem cells.

The putative existence of a cancer stem cell niche consisting of bi-directional stromal and stem cell secreting factors that trigger cancer stem cell growth and proliferation has been hypothesized in the nervous and hematopoietic systems. In light of this theory, it has been proposed that embryonic stem cell microenvironments, upon interactions with cancer stem cells, may reprogram cancer cells resulting in a substantial inhibition of tumor cell properties. Here, we discuss emerging data that support this novel concept of cancer inhibitory factors produced in the context of embryonic microenvironments as well as by embryonic stem cells (ESCs).