Among hematological cancers, Acute Lymphoblastic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL) are the most common leukemia in children and elderly people respectively. Some patients do not respond to chemotherapy treatments and it is necessary to complement it with immunotherapy-based treatments such as chimeric antigen receptor (CAR) therapy, which is one of the newest and more effective treatments against these cancers and B-cell lymphoma. Although complete remission results are promising, CAR T cell therapy presents still some risks for the patients, including cytokine release syndrome (CRS) and neurotoxicity. We proposed a different immune cell source for CAR therapy that might prevent these side effects while efficiently targeting malignant cells. NK cells from different sources are a promising vehicle for CAR therapy, as they do not cause graft versus host disease (GvHD) in allogenic therapies and they are prompt to attack cancer cells without prior sensitization. We studied the efficacy of NK cells from adult peripheral blood (AB) and umbilical cord blood (CB) against different target cells in order to determine the best source for CAR therapy. AB CAR-NK cells are slightly better at killing CD19 presenting target cells and CB NK cells are easier to stimulate and they have more stable number from donor to donor. We conclude that CAR-NK cells from both sources have their advantages to be an alternative and safer candidate for CAR therapy.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients’ survival.

How did the field of stem cell research develop in the years following the derivation of the first human embryonic stem cell (hESC) line? Supported by the increasing number of clinical trials to date, significant technological advances in the past two decades have brought us ever closer to clinical therapies derived from pluripotent cells. Since their discovery 20 years ago, the use of human pluripotent stem cells has progressed tremendously from bench to bedside. Here, we provide a concise review of the main keystones of this journey and focus on ongoing clinical trials, while indicating the most relevant future research directions. This is a historical narrative, including relevant publications in the field of pluripotent stem cells (PSC) derivation and differentiation, recounted both through scholarly research of published evidence and interviews of six pioneers who participated in some of the most relevant discoveries in the field. The authors all contributed by researching the literature and agreed upon body of works. Portions of the interviews of the field pioneers have been integrated into the review and have also been included in full for advanced reader interest. The stem cell field is ever expanding. We find that in the 20 years since the derivation of the first hESC lines, several relevant developments have shaped the pluripotent cell field, from the discovery of different states of pluripotency, the derivation of induced PSC, the refinement of differentiation protocols with several clinical trials underway, as well as the recent development of organoids. The challenge for the years to come will be to validate and refine PSCs for clinical use, from the production of highly defined cell populations in clinical grade conditions to the possibility of creating replacement organoids for functional, if not anatomical, function restoration. This is a non-systematic review of current literature. Some references may have escaped the experts’ analysis due to the exceedingly diverse nature of the field. As the field of regenerative medicine is rapidly advancing, some of the most recent developments may have not been captured entirely. The multi-disciplinary nature and tremendous potential of the stem cell field has important implications for basic as well as translational research. Recounting these activities will serve to provide an in-depth overview of the field, fostering a further understanding of human stem cell and developmental biology. The comprehensive overview of clinical trials and expert opinions included in this narrative may serve as a valuable scientific resource, supporting future efforts in translational approaches. ESHRE provided funding for the authors’ on-site meeting and discussion during the preparation of this manuscript. S.M.C.S.L. is funded by the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). M.G. is supported by the Methusalem grant of Vrije Universiteit Brussel, in the name of Prof. Karen Sermon and by Innovation by Science and Technology in Flanders (IWT, Project Number: 150042). A.V. and B.A. are supported by the Plataforma de Proteomica, Genotipado y Líneas Celulares (PT1770019/0015) (PRB3), Instituto de Salud Carlos III. Research grant to B.H. by the Research Foundation—Flanders (FWO) (FWO.KAN.2016.0005.01 and FWO.Project G051516N). There are no conflicts of interest to declare. Not applicable. ESHRE Pages are not externally peer reviewed. This article has been approved by the Executive Committee of 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.

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).

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSC), mainly because UCB is one of the most accessible HSC sources. In our study we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4-5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point towards the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.