Optimizing in vitro T cell differentiation by using induced pluripotent stem cells with GFP-RUNX1 and mCherry-TCF7 labelling.

In vitro T-cell differentiation from pluripotent stem cells (PSCs) could potentially provide an unlimited source of T cells for cancer immunotherapy, which, however is still hindered by the inefficient obtaining functionally-matured, terminally-differentiated T cells. Here, we established a fluorescence reporter human induced pluripotent stem cell (iPSC) line termed TCF7mCherryRUNX1GFP, in which the endogenous expression of RUNX1 and TCF7 are illustrated by the GFP and mCherry fluorescence, respectively. Utilizing TCF7mCherryRUNX1GFP, we defined that the feeder cells incorporating CXCL12-expressing OP9 cells with DL4-expressing OP9 cells at a 1:3 ratio (OP9-C1D3) significantly enhanced efficiency of CD8+ T cell differentiation from PSCs. Additionally, we engineered a chimeric antigen receptor (CAR) targeting EGFR into iPSCs. The CAR-T cells differentiated from these iPSCs using OP9-C1D3 feeders demonstrated effective cytotoxicity toward lung cancer cells. We anticipate this platform will help the in vitro HSPC and T cell differentiation optimization, serving the clinical demands of these cells.

Human midbrain dopaminergic progenitors.

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.

Technical specifications for ethics review of human stem cell research.

The rapid advancement of human stem cell research and its expansion into emerging areas has resulted in an escalation of ethical challenges associated with these studies. As a result, there has been a corresponding increase in both the volume and complexity of institutional ethics reviews, coupled with higher expectations for the quality of the review process. In response to these challenges, this standard provides a comprehensive outline of the fundamental principles, content, types, and procedures of ethics review, specifically focusing on non-clinical human stem cell research. Its purpose is to provide clear operational and procedural guidelines, as well as recommendations, for the ethics review of such studies. The document was originally published by the Chinese Society for Cell Biology on August 30, 2022. It is our hope that the publication of these guidelines will facilitate the integration of ethical considerations and evaluations in a structured manner throughout the entire process of stem cell research, ultimately fostering a healthy and orderly development of the field.

Human neural stem cells.

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

General requirements for the production of extracellular vesicles derived from human stem cells.

'General requirements for the production of extracellular vesicles derived from human stem cells' is the first guideline for stem cells derived extracellular vesicles in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the general requirements, process requirements, packaging and labelling requirements and storage requirements for preparing extracellular vesicles derived from human stem cells, which is applicable to the research and production of extracellular vesicles derived from stem cells. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardisation of extracellular vesicles derived from human stem cells.

Requirements for human natural killer cells.

'Requirements for Human Natural Killer Cells' is the latest set of guidelines on human NK cells in China, jointly drafted and agreed upon by experts from the Standards Committee of Chinese Society for Cell Biology. This standard specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells. This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 30 August, 2022. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.

Standard: Human intestinal cancer organoids.

Intestinal cancer is one of the most frequent and lethal types of cancer. Modeling intestinal cancer using organoids has emerged in the last decade. Human intestinal cancer organoids are physiologically relevant in vitro models, which provides an unprecedented opportunity for fundamental and applied research in colorectal cancer. "Human intestinal cancer organoids" is the first set of guidelines on human intestinal organoids in China, jointly drafted and agreed by the experts from the Chinese Society for Cell Biology and its branch society: the Chinese Society for Stem Cell Research. This standard specifies terms and definitions, technical requirements, test methods for human intestinal cancer organoids, which apply to the production and quality control during the process of manufacturing and testing of human intestinal cancer organoids. It was released by the Chinese Society for Cell Biology on 24 September 2022. We hope that the publication of this standard will guide institutional establishment, acceptance and execution of proper practocal protocols, and accelerate the international standardization of human intestinal cancer organoids for clinical development and therapeutic applications.

Standard: Human intestinal organoids.

Organoids have attracted great interest for disease modelling, drug discovery and development, and tissue growth and homeostasis investigations. However, lack of standards for quality control has become a prominent obstacle to limit their translation into clinic and other applications. "Human intestinal organoids" is the first guideline on human intestinal organoids in China, jointly drafted and agreed by the experts from the Chinese Society for Cell Biology and its branch society: the Chinese Society for Stem Cell Research. This standard specifies terms and definitions, technical requirements, test methods, inspection rules for human intestinal organoids, which is applicable to quality control during the process of manufacturing and testing of human intestinal organoids. It was originally released by the Chinese Society for Cell Biology on 24 September 2022. We hope that the publication of this standard will guide institutional establishment, acceptance and execution of proper practical protocols and accelerate the international standardization of human intestinal organoids for applications.

A novel inducible haematopoietic cell-depleting mouse model for chimeric complementation of blood cells.

Haematopoietic stem cell transplantation (HSCT) is widely used in regenerative medicine. HSCT can be used not only to treat certain types of blood cancer and immune disorders but also to induce immune tolerance in organ transplantation. However, the inadequacy of HSCs available for transplantation is still a major hurdle for clinical applications. Here, we established a novel inducible haematopoietic cell-depleting mouse model and tested the feasibility of using chimeric complementation to regenerate HSCs and their progeny cells. Large populations of syngeneic and major histocompatibility-mismatched haematopoietic cells were successfully regenerated by this model. The stable allogeneic chimeric mice maintained a substantial population of donor HSCs and Tregs, which indicated that the donor allogeneic HSCs successfully repopulated the recipient blood system, and the regenerated donor Tregs played essential roles in establishing immune tolerance in the allogeneic recipients. In addition, rat blood cells were detected in this model after xenotransplantation of rat whole bone marrow (BM) or Lin- BM cells. This mouse model holds promise for regenerating xenogeneic blood cells, including human haematopoietic cells.

Lin- PU.1dim GATA-1- defines haematopoietic stem cells with long-term multilineage reconstitution activity.

Despite extensive characterization of the state and function of haematopoietic stem cells (HSCs), the use of transcription factors to define the HSC population is still limited. We show here that the HSC population in mouse bone marrow can be defined by the distinct expression levels of Spi1 and Gata1. By using a double fluorescence knock-in mouse model, PGdKI, in which the expression levels of PU.1 and GATA-1 are indicated by the expression of GFP and mCherry, respectively, we uncover that the HSCs with lymphoid and myeloid repopulating activity are specifically enriched in a Lin- PU.1dim GATA-1- (LPG) cell subset. In vivo competitive repopulation assays demonstrate that bone marrow cells gated by LPG exhibit haematopoietic reconstitution activity which is comparable to that of classical Lin- Sca1+ c-kit+ (LSK). The integrated analysis of single-cell RNA sequence data from LPG and LSK-gated cells reveals that a transcriptional network governed by core TFs contributes to regulation of HSC multipotency. These discoveries provide new clues for HSC characterization and functional study.

Biomarkers of aging.

Aging biomarkers are a combination of biological parameters to (i) assess age-related changes, (ii) track the physiological aging process, and (iii) predict the transition into a pathological status. Although a broad spectrum of aging biomarkers has been developed, their potential uses and limitations remain poorly characterized. An immediate goal of biomarkers is to help us answer the following three fundamental questions in aging research: How old are we? Why do we get old? And how can we age slower? This review aims to address this need. Here, we summarize our current knowledge of biomarkers developed for cellular, organ, and organismal levels of aging, comprising six pillars: physiological characteristics, medical imaging, histological features, cellular alterations, molecular changes, and secretory factors. To fulfill all these requisites, we propose that aging biomarkers should qualify for being specific, systemic, and clinically relevant.

BNIP3-dependent mitophagy safeguards ESC genomic integrity via preventing oxidative stress-induced DNA damage and protecting homologous recombination.

Embryonic stem cells (ESCs) have a significantly lower mutation load compared to somatic cells, but the mechanisms that guard genomic integrity in ESCs remain largely unknown. Here we show that BNIP3-dependent mitophagy protects genomic integrity in mouse ESCs. Deletion of Bnip3 increases cellular reactive oxygen species (ROS) and decreases ATP generation. Increased ROS in Bnip3-/- ESCs compromised self-renewal and were partially rescued by either NAC treatment or p53 depletion. The decreased cellular ATP in Bnip3-/- ESCs induced AMPK activation and deteriorated homologous recombination, leading to elevated mutation load during long-term propagation. Whereas activation of AMPK in X-ray-treated Bnip3+/+ ESCs dramatically ascended mutation rates, inactivation of AMPK in Bnip3-/- ESCs under X-ray stress remarkably decreased the mutation load. In addition, enhancement of BNIP3-dependent mitophagy during reprogramming markedly decreased mutation accumulation in established iPSCs. In conclusion, we demonstrated a novel pathway in which BNIP3-dependent mitophagy safeguards ESC genomic stability, and that could potentially be targeted to improve pluripotent stem cell genomic integrity for regenerative medicine.

Evaluating mitophagy in embryonic stem cells by using fluorescence-based imaging.

Embryonic stem cells (ESCs), which are characterized by the capacity for self-renewal and pluripotency, hold great promise for regenerative medicine. Increasing evidence points to the essential role of mitophagy in pluripotency regulation. Our recent work showed that PINK1/OPTN take part in guarding ESC mitochondrial homeostasis and pluripotency. Evaluating mitophagy in ESCs is important for exploring the relationships between mitochondrial homeostasis and pluripotency. ESCs are smaller in size than adult somatic cells and the mitophagosomes in ESCs are difficult to observe. Many methods have been employed-for example, detecting colocalization of LC3-II and mitochondria-to evaluate mitophagy in ESCs. However, it is important to define an objective way to detect mitophagy in ESCs. Here, we evaluated two commonly used fluorescence-based imaging methods to detect mitophagy in ESCs. By using autophagy- or mitophagy-defective ESC lines, we showed that the mito-Keima (mt-Keima) system is a suitable and effective way for detecting and quantifying mitophagy in ESCs. Our study provides evidence that mt-Keima is an effective tool to study mitophagy function in ESCs.

Requirements for human-induced pluripotent stem cells.

'Requirements for Human-Induced Pluripotent Stem Cells' is the first set of guidelines on human-induced pluripotent stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, and instructions for use, labeling, packaging, storage, transportation, and waste handling for human-induced pluripotent stem cells, which apply to the production and quality control of human-induced pluripotent stem cells. It was released by the Chinese Society for Cell Biology on 9 January 2021 and came into effect on 9 April 2021. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human-induced pluripotent stem cells for applications.

Human retinal pigment epithelial cells.

'Human retinal pigment epithelial cells' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements and waste disposal requirements for human retinal pigment epithelial cells, which is applicable to quality control during the process of manufacturing and testing of human retinal pigment epithelial cells. It was originally released by the Chinese Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of human retinal pigment epithelial cells for applications.

Requirments for primary human hepatocyte.

'Requirements for Primary Human Hepatocyte' is the first set of guidelines on Primary Human Hepatocyte in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for Primary Human Hepatocyte, which is applicable to the quality control for Primary Human Hepatocyte. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of Primary Human Hepatocyte for applications.

Requirements for human haematopoietic stem/progenitor cells.

'Requirements for human haematopoietic stem/progenitor cells' is the first set of guidelines on human haematopoietic stem/progenitor cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, inspection methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements for human haematopoietic stem/progenitor cells, which is applicable to the quality control for human haematopoietic stem/progenitor cells. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human haematopoietic stem/progenitor cells for applications.

Human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have attracted great interest for cell therapy and tissue regeneration due to their self-renewal capacity, multipotency and potent immunomodulatory effects on immune cells. However, heterogeneity of MSCs has become a prominent obstacle to limit their translation into practice, as cells from different tissue sources or each individual have great differences in their transcriptomic signatures, differentiation potential and biological functions. Therefore, there is an urgent need for consensus standard for the quality control and technical specifications of MSCs. 'Human Mesenchymal Stem Cells' is the latest set of guidelines on hMSC in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hMSC, which is applicable to the quality control for hMSC. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hMSC for clinical development and therapeutic applications.