Cristina Eguizabal

Bioresorbable nanopatterned scaffolds functionalized with polydopamine (PDA) and graphene oxide (GO) have been shown to promote the differentiation of murine neural stem cells (mNSCs) toward neural and glial lineages. Herein, we aim to evaluate the compatibility of these scaffolds for the culture and differentiation of both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Our results indicate that PDA and GO scaffolds support the topographic alignment of hESCs and hiPSCs cultures, while preserving their pluripotency characteristics. Upon differentiation, PDA and GO scaffolds guide cell specification toward the neuroectoderm germ layer and the neural crest. This promotes enhanced differentiation into both neural and supportive glial cells of the central nervous system (CNS), as well as Schwann cells of the peripheral nervous system (PNS). Moreover, nanopatterned scaffolds also support the differentiation of hESCs and hiPSCs toward endothelial precursors. These findings establish a novel culture platform that enables combined differentiation pathways, potentially relevant for applications in personalized medicine and regenerative cell therapy.

Among adoptive cell-based immunotherapies, chimeric antigen receptor (CAR) therapy has shown promising results in cancer treatment. Treatment with CAR-T cells has produced remarkable clinical responses, especially in cases of relapsed and refractory leukemia and lymphoma. However, CAR-T cell therapy still presents several limitations, including some safety concerns related to neurotoxicity and aggressive inflammatory responses. As an alternative to T-cells, natural killer (NK) cells have been developed as an attractive option for efficient cancer immunotherapy. As they do not express T cell receptor (TCR), NK cell-based therapies are not associated with cytokine release syndrome (CRS) or graft versus host disease (GvHD), which enables a safer therapy and the ability to generate an allogeneic "off-the-shelf" product. Despite the innate cytotoxic activity of NK cells against malignant cells, the therapeutic application of unmodified NK cells has been compromised by the inhibitory tumor microenvironment (TME), which is responsible for poor cell expansion, inactivation, insufficient tumor infiltration, and limited in vivo persistence, leading to the dysfunction of NK cells after infusion. Advances in the genetic modification of NK cells can address some of these limitations and improve their therapeutic efficacy. In this review, we describe the advances in the development of engineered NK cells for cancer immunotherapy. As such, we provide an overview of recent viral and non-viral approaches for the genetic modification of NK cells. We also discuss their current clinical status in the field of immunotherapy, and their use in other clinical applications.

STUDY QUESTION: How should fertility preservation in child and adolescent males receiving gonadotoxic therapies be managed? SUMMARY ANSWER: There were 44 recommendations formulated to provide guidance on all aspects of fertility preservation in prepubertal boys and adolescent males in whom it is not possible to obtain sperm by established methods including semen cryopreservation and surgical sperm extraction; the recommendations covered topics from setting up a fertility preservation program, determining who is eligible, and counselling, to the practical aspects of the testicular tissue biopsy and cryopreservation. WHAT IS KNOWN ALREADY: For young males facing gonadotoxic treatment, there are limited options for fertility preservation. For those who are unable to produce sperm (children and adolescents) testicular tissue cryopreservation is being increasingly offered prior to gonadotoxic treatment for potential future clinical use to restore fertility. STUDY DESIGN, SIZE, DURATION: This Good Practice Recommendations (GPR) article was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, if available. PARTICIPANTS/MATERIALS, SETTING, METHODS: ESHRE appointed a European multidisciplinary working group with expertise in paediatric oncology, paediatric endocrinology, human tissue banking, and surgery, as well as reproductive specialists and researchers, who have demonstrated leadership and expertise in fertility preservation in prepubertal boys and adolescent males. The recommendations were formulated based on the expert opinion of the working group, while taking into consideration the published data. The draft document was then open to ESHRE members for online peer review and was revised in light of the comments received. MAIN RESULTS AND THE ROLE OF CHANCE: The multidisciplinary working group formulated 44 recommendations to provide guidance on all aspects of fertility preservation by testicular tissue cryopreservation, from setting up a fertility preservation program, determining who is eligible and counselling, to the practical aspects of the testicular tissue biopsy and cryopreservation. LIMITATIONS, REASON FOR CAUTION: These guidelines offer valuable direction for healthcare professionals taking care of children and adolescents facing gonadotoxic treatment. Their purpose is to promote knowledge among clinicians and to enable patients to make informed decisions based on realistic expectations. WIDER IMPLICATIONS OF THE FINDINGS: This article provides not only good practice advice but also highlights the areas that need further research. This research, when well-conducted, will be key to making progress in the area of fertility preservation in children and adolescents. STUDY FUNDING/COMPETING INTERESTS: All costs relating to the development process, including meetings and technical support for this project, were funded by the European Society of Human Reproduction and Embryology (ESHRE). There was no external funding of the development process or manuscript production. RTM was supported by a UKRI Future Leader Fellowship. The other authors disclosed no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A. DISCLAIMER: This Good Practice Recommendations document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

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? 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. 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. 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. 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. 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. 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. 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. 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. N/A.

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.

Study question: Can we accurately assess sex-chromosomes number in testicular cells (germ and somatic) of Klinefelter Syndrome (KS) prepuberal patients by DNA-FISH in paraffin embedded sections? Summary answer: We have evaluated the sex-chromosomes content of testicular cells in KS prepuberal patients and the presence of XY lines evidenced a gonadal mosaicism status. What is known already: Prepuberal boys with Klinefelter Syndrome usually have fertility problems in adulthood, mainly azoospermia. Due to the symptoms cause by the disease, Spermatogonial Stem Cells (SSCs) are less abundant and spermatogenesis does not occur. These patients do not have any alternative to restore the fertility in the future. When a boy has both XY and XXY cell lines, this patient is mosaic (46,XY/47,XXY). Meaning some cells could undergo spermatogenesis and generate gametes with normal sex-chromosomes number. The aim of this work is to test if KS patients diagnosed as pure can have testicular cell lines with XY chromosome number (mosaic). Study design, size, duration: We obtained samples from human prepuberal patients diagnosed with pure Klinefelter Syndrome (47,XXY) for the last 5 years. Testicular biopsy fragments are fixed for histological studies and other fragments are cryopreserved. Some fragments are used for immunofluorescence and subsequently processed by DNA-FISH to determine the sex-chromosomes content of testicular cells. Participants/materials, setting, methods: In this study, we used 10 prepuberal patients with KS and 5 prepuberal patients with other fertility problems with normal chromosome set as controls. We performed immunofluorescence to determine expression of germ cells (VASA) and SSCs (MAGEA4) markers and somatic cells markers such as Leydig cells (StAR) and Sertoli cells (SOX9). Afterwards, we perform DNA-FISH, with probes specific for chromosomes X and Y and chromosome 18 as a control. Main results and the role of chance: The methodology used allows cytogenetic characterization of testicular tissue in paraffin embedded sections. Testicular mosaicism has been observed in all patients diagnosed as pure KS. We have observed a degree of mosaicism of 66-80% in SSCs, of 20-50% in Sertoli cells and of 30-50% in Leydig cells. We pursued a protocol with a good FISH efficiency that allows colocalization of previous immunocharacterized testicular cells. Limitations, reasons for caution: The DNA-FISH technique is made just in a single paraffin section of each KS patient testicular sample. Due to the limited and difficulty to obtain KS testicular tissue, we use a limited number of samples. Wider implications of the findings: We succeed to demonstrate the mosaicism of testicular cells in prepuberal patients that are diagnosed with KS. Thus, the better understanding of the SSCs with normal chromosome set (XY) could be useful for future in vitro expansion and stem cell therapies. Trial registration number: not applicable

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.

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).