Results of phase 2 randomized multi-center study to evaluate the safety and efficacy of infusion of memory T cells as adoptive therapy in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and/or lymphopenia (RELEASE NCT04578210).

BACKGROUND AIMS: There are currently no effective anti-viral treatments for coronavirus disease 2019 (COVID-19)-hospitalized patients with hypoxemia. Lymphopenia is a biomarker of disease severity usually present in patients who are hospitalized. Approaches to increasing lymphocytes exerting an anti-viral effect must be considered to treat these patients. Following our phase 1 study, we performed a phase 2 randomized multicenter clinical trial in which we evaluated the efficacy of the infusion of allogeneic off-the-shelf CD45RA- memory T cells containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells from convalescent donors plus the standard of care (SoC) versus just the SoC treatment. METHODS: Eighty-four patients were enrolled in three Spanish centers. The patients were randomized into the infusion of 1 × 106/kg CD45RA- memory T cells or the SoC. We selected four unvaccinated donors based on the expression of interferon gamma SARS-CoV-2-specific response within the CD45RA- memory T cells and the most frequent human leukocyte antigen typing in the Spanish population. RESULTS: We analyzed data from 81 patients. The primary outcome for recovery, defined as the proportion of participants in each group with normalization of fever, oxygen saturation sustained for at least 24 hours and lymphopenia recovery through day 14 or at discharge, was met for the experimental arm. We also observed faster lymphocyte recovery in the experimental group. We did not observe any treatment-related adverse events. CONCLUSIONS: Adoptive cell therapy with off-the-shelf CD45RA- memory T cells containing SAR-CoV-2-specific T cells is safe, effective and accelerates lymphocyte recovery of patients with COVID-19 pneumonia and/or lymphopenia. TRIAL REGISTRATION: NCT04578210.

Transcriptional progression during meiotic prophase I reveals sex-specific features and X chromosome dynamics in human fetal female germline.

During gametogenesis in mammals, meiosis ensures the production of haploid gametes. The timing and length of meiosis to produce female and male gametes differ considerably. In contrast to males, meiotic prophase I in females initiates during development. Hence, the knowledge regarding progression through meiotic prophase I is mainly focused on human male spermatogenesis and female oocyte maturation during adulthood. Therefore, it remains unclear how the different stages of meiotic prophase I between human oogenesis and spermatogenesis compare. Analysis of single-cell transcriptomics data from human fetal germ cells (FGC) allowed us to identify the molecular signatures of female meiotic prophase I stages leptotene, zygotene, pachytene and diplotene. We have compared those between male and female germ cells in similar stages of meiotic prophase I and revealed conserved and specific features between sexes. We identified not only key players involved in the process of meiosis, but also highlighted the molecular components that could be responsible for changes in cellular morphology that occur during this developmental period, when the female FGC acquire their typical (sex-specific) oocyte shape as well as sex-differences in the regulation of DNA methylation. Analysis of X-linked expression between sexes during meiotic prophase I suggested a transient X-linked enrichment during female pachytene, that contrasts with the meiotic sex chromosome inactivation in males. Our study of the events that take place during meiotic prophase I provide a better understanding not only of female meiosis during development, but also highlights biomarkers that can be used to study infertility and offers insights in germline sex dimorphism in humans.

Predicting Optimal Antimalarial Drug Combinations from a Standardized Plasmodium falciparum Humanized Mouse Model.

The development of new combinations of antimalarial drugs is urgently needed to prevent the spread of parasites resistant to drugs in clinical use and contribute to the control and eradication of malaria. In this work, we evaluated a standardized humanized mouse model of erythrocyte asexual stages of Plasmodium falciparum (PfalcHuMouse) for the selection of optimal drug combinations. First, we showed that the replication of P. falciparum was robust and highly reproducible in the PfalcHuMouse model by retrospective analysis of historical data. Second, we compared the relative value of parasite clearance from blood, parasite regrowth after suboptimal treatment (recrudescence), and cure as variables of therapeutic response to measure the contributions of partner drugs to combinations in vivo. To address the comparison, we first formalized and validated the day of recrudescence (DoR) as a new variable and found that there was a log-linear relationship with the number of viable parasites per mouse. Then, using historical data on monotherapy and two small cohorts of PfalcHuMice evaluated with ferroquine plus artefenomel or piperaquine plus artefenomel, we found that only measurements of parasite killing (i.e., cure of mice) as a function of drug exposure in blood allowed direct estimation of the individual drug contribution to efficacy by using multivariate statistical modeling and intuitive graphic displays. Overall, the analysis of parasite killing in the PfalcHuMouse model is a unique and robust experimental in vivo tool to inform the selection of optimal combinations by pharmacometric pharmacokinetic and pharmacodynamic (PK/PD) modeling.

Natural killer cell subsets in endometrial fluid: a pilot study of their association with the endometrial cycle and reproductive parameters.

PURPOSE: To investigate if there are natural killer (NK) cells in endometrial fluid (EF) and their relationship with the endometrial cycle and reproductive parameters. METHODS: The population under study consisted of 43 women aged 18-40 undergoing infertility workup at our University Hospital in 2021-2022. The EF samples were obtained at the first visit to our unit, on occasion of the mock embryo transfer. The day of the cycle was considered only in cycles of 27-29 days. An immunophenotype study of NK in EF was performed by flow cytometry analysis. In a subgroup of women, on the same day, NK was studied in EF and peripheral blood. RESULTS: Our study is the first to evidence NK cells in EF. None of the NK cells observed corresponded to a mature peripheral blood NK cell population (stages 4-5), and neither endometrial nor decidual uNK cells were detected. Nevertheless, we found 2 patient groups with an NK cell subset with a higher expression of CD16+, which could belong to an intermediate or transient stage between the uNK and pbNK NK cell population in the EF. We found that CD16 was significantly increased in the mid-late luteal phase and its correlation with the day of the cycle. The NK immunophenotype was different in EF and peripheral blood. CONCLUSION: We described a new component of the EF, the NK cells, whose CD16 activity is closely correlated with the day of the cycle. These cells could play a role in implantation/implantation failure.

Identifying SARS-CoV-2 'memory' NK cells from COVID-19 convalescent donors for adoptive cell therapy.

COVID-19 disease is the manifestation of syndrome coronavirus 2 (SARS-CoV-2) infection, which is causing a worldwide pandemic. This disease can lead to multiple and different symptoms, being lymphopenia associated with severity one of the most persistent. Natural killer cells (NK cells) are part of the innate immune system, being fighting against virus-infected cells one of their key roles. In this study, we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2-specific-like NK population in the blood of convalescent donors. CD57+ NKG2C+ phenotype in SARS-CoV-2 convalescent donors indicates the presence of 'memory'/activated NK cells as it has been shown for cytomegalovirus infections. Although the existence of this population is donor dependent, its expression may be crucial for the specific response against SARS-CoV-2, so that, it gives us a tool for selecting the best donors to produce off-the-shelf living drug for cell therapy to treat COVID-19 patients under the RELEASE clinical trial (NCT04578210).

Uneven metabolic and lipidomic profiles in recovered COVID-19 patients as investigated by plasma NMR metabolomics.

COVID-19 is a systemic infectious disease that may affect many organs, accompanied by a measurable metabolic dysregulation. The disease is also associated with significant mortality, particularly among the elderly, patients with comorbidities, and solid organ transplant recipients. Yet, the largest segment of the patient population is asymptomatic, and most other patients develop mild to moderate symptoms after SARS-CoV-2 infection. Here, we have used NMR metabolomics to characterize plasma samples from a cohort of the abovementioned group of COVID-19 patients (n = 69), between 3 and 10 months after diagnosis, and compared them with a set of reference samples from individuals never infected by the virus (n = 71). Our results indicate that half of the patient population show abnormal metabolism including porphyrin levels and altered lipoprotein profiles six months after the infection, while the other half show little molecular record of the disease. Remarkably, most of these patients are asymptomatic or mild COVID-19 patients, and we hypothesize that this is due to a metabolic reflection of the immune response stress.

How donor selection criteria can be evaluated with limited scientific evidence: lessons learned from the TRANSPOSE project.

BACKGROUND AND OBJECTIVE: Donor selection criteria (DSC) are a vital link in the chain of supply of Substances of Human Origin (SoHO) but are also subject to controversy and differences of opinion. Traditionally, DSC have been based on application of the precautionary principle. MATERIALS AND METHODS: From 2017 to 2020, TRANSPOSE (TRANSfusion and transplantation PrOtection and SElection of donors), a European research project, aimed to identify discrepancies between current DSC by proposing a standardized risk assessment method for all SoHO (solid organs excluded) and all levels of evidence. RESULTS: The current DSC were assessed using a modified risk assessment method based on the Alliance of Blood Operators' Risk-based decision-making framework for blood safety. It was found that with limited or diverging scientific evidence, it was difficult to reach consensus and an international standardized method for decision-making was lacking. Furthermore, participants found it hard to disregard their local guidelines when providing expert opinion, which resulted in substantial influence on the consensus-based decision-making process. CONCLUSIONS: While the field of donation-safety research is expanding rapidly, there is an urgent need to formalize the decision-making process regarding DSC. This includes the need for standardized methods to increase transparency in the international decision-making process and to ensure that this is performed consistently. Our framework provides an easy-to-implement approach for standardizing risk assessments, especially in the context of limited scientific evidence.

Putting the spotlight on donation-related risks and donor safety - are we succeeding in protecting donors?

BACKGROUND AND OBJECTIVE: The European consortium project TRANSPOSE (TRANSfusion and transplantation: PrOtection and SElection of donors) aimed to assess and evaluate the risks to donors of Substances of Human Origin (SoHO), and to identify gaps between current donor vigilance systems and perceived risks. MATERIALS AND METHODS: National and local data from participating organizations on serious and non-serious adverse reactions in donors were collected from 2014 to 2017. Following this, a survey was performed among participants to identify risks not included in the data sets. Finally, participants rated the risks according to severity, level of evidence and prevalence. RESULTS: Significant discrepancies between anticipated donor risks and the collected data were found. Furthermore, many participants reported that national data on adverse reactions in donors of stem cells, gametes, embryos and tissues were not routinely collected and/or available. CONCLUSIONS: These findings indicate that there is a need to further develop and standardize donor vigilance in Europe and to include long-term risks to donors, which are currently underreported, ensuring donor health and securing the future supply of SoHO.

Human-specific subcellular compartmentalization of P-element induced wimpy testis-like (PIWIL) granules during germ cell development and spermatogenesis.

STUDY QUESTION: What is the dynamics of expression of P-element induced wimpy testis-like (PIWIL) proteins in the germline during human fetal development and spermatogenesis? SUMMARY ANSWER: PIWIL1, PIWIL2, PIWIL3 and PIWIL4 were expressed in a sex-specific fashion in human germ cells (GC) during development and adulthood. PIWILs showed a mutually exclusive pattern of subcellular localization. PIWILs were present in the intermitochondrial cement and a single large granule in meiotic GC and their expression was different from that observed in mice, highlighting species-differences. WHAT IS KNOWN ALREADY: In mice, PIWIL proteins play prominent roles in male infertility. PIWIL mouse mutants show either post-meiotic arrest at the round spermatid stage (PIWIL1) or arrest at the zygotene-pachytene stage of meiosis I (PIWIL2 and PIWIL4) in males, while females remain fertile. Recent studies have reported a robust piRNA pool in human fetal ovary. STUDY DESIGN, SIZE, DURATION: This is a qualitative analysis of PIWILs expression in paraffin-embedded fetal human male (N = 8), female gonads (N = 6) and adult testes (N = 5), and bioinformatics analysis of online available single-cell transcriptomics data of human fetal germ cells (n = 242). PARTICIPANTS/MATERIALS, SETTING, METHODS: Human fetal gonads from elective abortion without medical indication and adult testes biopsies were donated for research with informed consent. Samples were fixed, paraffin-embedded and analyzed by immunofluorescence to study the temporal and cellular localization of PIWIL1, PIWIL2, PIWIL3 and PIWIL4. MAIN RESULTS AND THE ROLE OF CHANCE: PIWIL1, PIWIL2 and PIWIL4 showed a mutually exclusive pattern of subcellular localization, particularly in female oocytes. To our surprise, PIWIL1 immunostaining revealed the presence of a single dense paranuclear body, resembling the chromatoid body of haploid spermatocytes, in meiotic oocytes. Moreover, in contrast to mice, PIWIL4, but not PIWIL2, localized to the intermitochondrial cement. PIWIL3 was not expressed in GC during development. The upregulation of PIWIL transcripts correlated with the transcription of markers associated with piRNAs biogenesis like the TDRDs and HENMT1 in fetal GC. LARGE SCALE DATA: Non-applicable. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the restricted number of samples and consequently stages analyzed. WIDER IMPLICATIONS OF THE FINDINGS: In the germline, PIWILs ensure the integrity of the human genome protecting it from 'parasitic sequences'. This study offers novel insights on the expression dynamics of PIWILs during the window of epigenetic remodeling and meiosis, and highlights important differences between humans and mice, which may prove particularly important to understand causes of infertility and improve both diagnosis and treatment in humans. STUDY FUNDING/COMPETING INTEREST(S): M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011]; N.H. by China Scholarship Council (CSC) [No. 201307040026] and F.W. by Medical Personnel Training Abroad Project of Henan Province [No. 2015022] and S.M.C.d.S.L. by the Netherlands Organization of Scientific Research (NWO) [ASPASIA 015.007.037] and the Interuniversity Attraction Poles-Phase VII [IUAP/PAI P7/14]. The authors have no conflicts of interest to declare.

The Biology and Disease Relevance of CD300a, an Inhibitory Receptor for Phosphatidylserine and Phosphatidylethanolamine.

The CD300a inhibitory receptor belongs to the CD300 family of cell surface molecules that regulate a diverse array of immune cell processes. The inhibitory signal of CD300a depends on the phosphorylation of tyrosine residues embedded in ITIMs of the cytoplasmic tail. CD300a is broadly expressed on myeloid and lymphoid cells, and its expression is differentially regulated depending on the cell type. The finding that CD300a recognizes phosphatidylserine and phosphatidylethanolamine, two aminophospholipids exposed on the outer leaflet of dead and activated cells, has shed new light on its role in the modulation of immune functions and in its participation in the host response to several diseases states, such as infectious diseases, cancer, allergy, and chronic inflammatory diseases. This review summarizes the literature on CD300a expression, regulation, signaling pathways, and ligand interaction, as well as its role in fine tuning immune cell functions and its clinical relevance.

Neuronopathic Gaucher's disease: induced pluripotent stem cells for disease modelling and testing chaperone activity of small compounds.

Gaucher's disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-ß-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-ß-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-ß-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.

Intracranial graft of bioresorbable polymer scaffolds loaded with human Dental Pulp Stem Cells in stab wound murine injury model.

The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization.

A 20-year overview of fertility preservation in boys: new insights gained through a comprehensive international survey.

STUDY QUESTION: Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER: Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY: For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN SIZE DURATION: A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS SETTING METHODS: Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6-20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS REASONS FOR CAUTION: While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS: Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTERESTS: K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation-Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children's Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G., N.N., S.S., C.L.M., A.M.M.v.P., C.E., R.T.M., K.D., M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Analysis of the impact of handling and culture on the expansion and functionality of NK cells.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a key role in the elimination of tumor and virus-infected cells. Unlike T cells, NK cell activation is governed by their direct interaction with target cells via the inhibitory and activating receptors present on their cytoplasmic membrane. The simplicity of this activation mechanism has allowed the development of immunotherapies based on the transduction of NK cells with CAR (chimeric antigen receptor) constructs for the treatment of cancer. Despite the advantages of CAR-NK therapy over CAR-T, including their inability to cause graft-versus-host disease in allogenic therapies, a deeper understanding of the impact of their handling is needed in order to increase their functionality and applicability. With that in mind, the present work critically examines the steps required for NK cell isolation, expansion and storage, and analyze the response of the NK cells to these manipulations. The results show that magnetic-assisted cell sorting, traditionally used for NK isolation, increases the CD16+ population of NK cultures only if the protocol includes both, antibody incubation and passage through the isolation column. Furthermore, based on the importance of surface potential on cellular responses, the influence of surfaces with different net surface charge on NK cells has been evaluated, showing that NK cells displayed higher proliferation rates on charged surfaces than on non-charged ones. The present work highlights the relevance of NK cells manipulation for improving the applicability and effectiveness of NK cell-based therapies.

Cell-cell interactions during the formation of primordial follicles in humans.

Gametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female gametogenesis is the formation of primordial follicles, the functional unit of the ovary that constitutes the pool of follicles available at birth during the entire reproductive life. However, the relation between human fetal germ cells (hFGCs) and gonadal somatic cells during the formation of the primordial follicles remains largely unexplored. We have discovered that hFGCs can form multinucleated syncytia, some connected via interconnecting intercellular bridges, and that not all nuclei in hFGC-syncytia were synchronous regarding meiotic stage. As hFGCs progressed in development, pre-granulosa cells formed protrusions that seemed to progressively constrict individual hFGCs, perhaps contributing to separate them from the multinucleated syncytia. Our findings highlighted the cell-cell interaction and molecular dynamics between hFGCs and (pre)granulosa cells during the formation of primordial follicles in humans. Knowledge on how the pool of primordial follicle is formed is important to understand human infertility.

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.

Simple generation of human induced pluripotent stem cells using poly-beta-amino esters as the non-viral gene delivery system.

Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-ß-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-ß-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-ß-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.

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.

Improving In Vitro Culture of Human Male Fetal Germ Cells.

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.