Transient characteristics of universal cells on human‐induced pluripotent stem cells and their differentiated cells derived from foetal stem cells with mixed donor sources

IntroductionIt is important to prepare ‘hypoimmunogenic’ or ‘universal’ human pluripotent stem cells (hPSCs) with gene‐editing technology by knocking out or in immune‐related genes, because only a few hypoimmunogenic or universal hPSC lines would be sufficient to store for their off‐the‐shelf use. However, these hypoimmunogenic or universal hPSCs prepared previously were all genetically edited, which makes laborious processes to check and evaluate no abnormal gene editing of hPSCs.MethodsUniversal human‐induced pluripotent stem cells (hiPSCs) were generated without gene editing, which were reprogrammed from foetal stem cells (human amniotic fluid stem cells) with mixing 2‐5 allogenic donors but not with single donor. We evaluated human leucocyte antigen (HLA)‐expressing class Ia and class II of our hiPSCs and their differentiated cells into embryoid bodies, cardiomyocytes and mesenchymal stem cells. We further evaluated immunogenic response of transient universal hiPSCs with allogenic mononuclear cells from survival rate and cytokine production, which were generated by the cells due to immunogenic reactions.ResultsOur universal hiPSCs during passages 10‐25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21‐22 survived and continued beating even after treatment with allogenic mononuclear cells.     

A CRMP4‐dependent retrograde axon‐to‐soma death signal in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal non‐cell‐autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1^G93A‐ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72‐mutant patients, and the SOD1^G93A‐ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS‐affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4‐dynein interaction reduces MN loss in human‐derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4‐dependent retrograde death signal that underlies MN loss in ALS.     

Cytochrome P450 Cyp26a1 Alters Spinal Motor Neuron Subtype Identity in Differentiating Embryonic Stem Cells

The ability to differentiate embryonic stem cells (ESCs) into specific cell types is critical for improved regenerative medicine strategies, cancer chemotherapeutic approaches, and regimens to combat chronic diseases associated with aging. Subclasses of motor neurons (MNs) are generated at different positions along the rostrocaudal axis of the spinal cord, and the signals that specify MN subtype fates remain poorly defined. We show here that the cytochrome P450 enzyme Cyp26a1, which metabolizes all-trans-retinoic acid (RA) and thereby reduces RA levels, plays a crucial role in specifying MN columnar subtypes. Lack of Cyp26a1 in ESCs during differentiation to spinal MNs increases Aldh1a2 (RALDH2) and Hoxc6, markers of the Hox-dependent, lateral motor column (LMC) subtype identity. In contrast, Lhx3, a marker for median motor column identity, showed lower expression in Cyp26a1^−/−-derived MNs compared with WT. Without Cyp26a1, an increase in intracellular RA concentration plus sonic hedgehog agonist treatment confer an LMC fate on differentiating MNs. Our data suggest a strategy for increasing LMC-type MNs from ESCs by blocking Cyp26a1 in cell replacement/ESC differentiation therapy to treat neurodegenerative diseases, such as amyotrophic lateral sclerosis.     

Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

ObjectivesThe rats are crucial animal models for the basic medical researches. Rat embryonic stem cells (ESCs), which are widely studied, can self‐renew and exhibit pluripotency in long‐term culture, but the mechanism underlying how they exit pluripotency remains obscure. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome‐wide screening using a unique rat haploid system.Materials and MethodsRat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1‐GFP reporter are used for genetic screening. Genome‐wide mutations are introduced into the genomes of rat Rex1‐GFP haESCs via piggyBac transposon, and differentiation‐retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high‐throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p‐ERK1/2) is determined by western blotting.ResultsHigh‐throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p‐ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to exit from pluripotency.ConclusionsOur findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self‐renewal or pluripotency of rat ESCs.

Differentiation of pluripotent rat embryonic stem cells (ESCs) in vitro is difficult to achieve for unknown mechanisms. Rat haploid ESCs (haESCs) have been validated as a powerful tool to target unknown functional genes and pathways based on homozygous genetic screening. Xu et al. utilized Rex1‐GFP labelled‐rat haESCs to conduct genome‐scale screening of genes modulating pluripotency exiting. Validation experiments showed that Thop1 (one of the screened out genes) played very important roles in the random differentiation of rat ESCs in vitro via modulating phosphorylation of ERK.     

Human iPSC‐derived neural precursor cells differentiate into multiple cell types to delay disease progression following transplantation into YAC128 Huntington's disease mouse model

ObjectivesTo investigate whether human HLA‐homozygous induced pluripotent stem cell (iPSC)‐derived neural precursor cells (iPSC‐NPCs) can provide functional benefits in Huntington’s disease (HD), we transplanted them into the YAC128 transgenic HD mouse model.Materials and MethodsCHAi001‐A, an HLA‐homozygous iPSC line (A*33:03‐B*44:03‐DRB1*13:02), was differentiated into neural precursor cells, and then, they were transplanted into 6 months‐old YAC128 mice. Various behavioural and histological analyses were performed for five months after transplantation.ResultsMotor and cognitive functions were significantly improved in transplanted animals. Cells transplanted in the striatum showed multipotential differentiation. Five months after transplantation, the donor cells had differentiated into neurons, oligodendrocytes and astrocytes. Transplantation restored DARPP‐32 expression, synaptophysin density, myelin basic protein expression in the corpus callosum and astrocyte function.ConclusionAltogether, these results strongly suggest that iPSC‐NPCs transplantation induces neuroprotection and functional recovery in a mouse model of HD and should be taken forward for clinical trials in HD patients.     

Selection of O‐negative induced pluripotent stem cell clones for high‐density red blood cell production in a scalable perfusion bioreactor system

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Human pluripotent stem cell‐derived red blood cells could help alleviate worldwide blood shortages and provide a secure year‐round supply. However, current generation methods lack the necessary scalability. Here, we present the selection of O‐negative hiPSC clones and demonstrate the small‐scale generation of functional erythroblasts in a high‐density perfusion bioreactor system.     

Ginsenoside Rh2 mitigates doxorubicin‐induced cardiotoxicity by inhibiting apoptotic and inflammatory damage and weakening pathological remodelling in breast cancer‐bearing mice

ObjectivesThere are presently a few viable ways to reduce cardiotoxicity of doxorubicin (Dox). The combination of chemotherapy agents with natural compounds delivers greater efficacy and reduces adverse effects in recent researches for cancer treatment. Here, we examined the potential effect of ginsenoside Rh2 on a Dox‐based regimen in chemotherapy treatment.Materials and MethodsHuman breast tumour (MDA‐MB‐231) xenograft nude mice, human cardiac ventricle fibroblasts, and human umbilical vein endothelial cells (HUVEC) were employed in the present study. Histology, immunohistochemistry, immunofluorescence, western blot, antibody array, and RNA‐sequencing analyses were utilized to assess the protective effect of Rh2 on cardiotoxicity induced by Dox and the underlying mechanisms.ResultsRh2‐reduced cardiotoxicity by inhibiting the cardiac histopathological changes, apoptosis and necrosis, and consequent inflammation. Pathological remodelling was attenuated by reducing fibroblast to myofibroblast transition (FMT) and endothelial–mesenchymal transition (EndMT) in hearts. RNA‐sequencing analysis showed that Dox treatment predominantly targets cell cycle and attachment of microtubules and boosted tumour necrosis, chemokine and interferon‐gamma production, response to cytokine and chemokine, and T cell activation, whereas Rh2 regulated these effects. Intriguingly, Rh2 also attenuated fibrosis via promoting senescence in myofibroblasts and reversing established myofibroblast differentiation in EndMT.ConclusionsRh2 regulates multiple pathways in the Dox‐provoked heart, proposing a potential candidate for cancer supplement and therapy‐associated cardiotoxicity.

Doxorubicin is extensively reported to induce severe cardiotoxicity in clinical applications. Our work proposed a natural herbal compound, ginsenoside Rh2, as a potential candidate for attenuating this side effect. Rh2 significantly inhibited cardiac apoptosis and necrosis, inflammation, and pathological remodelling in Dox‐challenged hearts.