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Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits, however, relies on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson's disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9-assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single-cell RNA sequencing of iPSC-derived neural cultures confirm developmental recapitulation of the human fetal midbrain and high-quality midbrain cells. By modelling Parkinson's disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP+), we showed a preferential reduction of BFP+ cells, a finding demonstrated independently by cell death assays and single-cell transcriptomic analysis of MPP+ treated neural cultures. Together, these results highlight the importance of disease-relevant cell types in stem cell modelling. [ABSTRACT FROM AUTHOR]

Red blood cell (RBC) transfusion is a lifesaving medical procedure that can treat patients with anemia and hemoglobin disorders. However, the shortage of blood supply and risks of transfusion-transmitted infection and immune incompatibility present a challenge for transfusion. The in vitro generation of RBCs or erythrocytes holds great promise for transfusion medicine and novel cell-based therapies. While hematopoietic stem cells and progenitors derived from peripheral blood, cord blood, and bone marrow can give rise to erythrocytes, the use of human pluripotent stem cells (hPSCs) has also provided an important opportunity to obtain erythrocytes. These hPSCs include both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). As hESCs carry ethical and political controversies, hiPSCs can be a more universal source for RBC generation. In this review, we first discuss the key concepts and mechanisms of erythropoiesis. Thereafter, we summarize different methodologies to differentiate hPSCs into erythrocytes with an emphasis on the key features of human definitive erythroid lineage cells. Finally, we address the current limitations and future directions of clinical applications using hiPSC-derived erythrocytes. [ABSTRACT FROM AUTHOR]

Heart diseases such as myocardial infarction and myocardial ischemia are paroxysmal and fatal in clinical practice. Cardiomyocytes (CMs) differentiated from human pluripotent stem cells provide a promising approach to myocardium regeneration therapy. Identifying the maturity level of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is currently the main challenge for pathophysiology and therapeutics. In this review, we describe current maturity indicators for cardiac microtissue and microdevice cultivation technologies that accelerate cardiac maturation. It may provide insights into regenerative medicine, drug cardiotoxicity testing, and preclinical safety testing. [ABSTRACT FROM AUTHOR]

GABAergic interneurons control the neural circuitry and network activity in the brain. The dysfunction of cortical interneurons, especially those derived from the medial ganglionic eminence, contributes to neurological disease states. Pluripotent stem cell-derived interneurons provide a powerful tool for understanding the etiology of neuropsychiatric disorders, as well as having the potential to be used as medicine in cell therapy for neurological conditions such as epilepsy. Although large numbers of interneuron progenitors can be readily induced in vitro, the generation of defined interneuron subtypes remains inefficient. Using CRISPR/Cas9-assisted homologous recombination in hPSCs, we inserted the coding sequence of mEmerald and mCherry fluorescence protein, respectively, downstream that of the LHX6, a gene required for, and a marker of medial ganglionic eminence (MGE)-derived cortical interneurons. Upon differentiation of the LHX6-mEmerald and LHX6-mCherry hPSCs towards the MGE fate, both reporters exhibited restricted expression in LHX6+ MGE derivatives of hPSCs. Moreover, the reporter expression responded to changes of interneuron inductive cues. Thus, the LHX6-reporter lines represent a valuable tool to identify molecules controlling human interneuron development and design better interneuron differentiation protocols as well as for studying risk genes associated with interneuronopathies. [ABSTRACT FROM AUTHOR]

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery. [ABSTRACT FROM AUTHOR]

Cataract is a leading cause of blindness worldwide. Currently, restoration of vision in cataract patients requires surgical removal of the cataract. Due to the large and increasing number of cataract patients, the annual cost of surgical cataract treatment amounts to billions of dollars. Limited access to functional human lens tissue during the early stages of cataract formation has hampered efforts to develop effective anti-cataract drugs. The ability of human pluripotent stem (PS) cells to make large numbers of normal or diseased human cell types raises the possibility that human PS cells may provide a new avenue for defining the molecular mechanisms responsible for different types of human cataract. Towards this end, methods have been established to differentiate human PS cells into both lens cells and transparent, light-focusing human micro-lenses. Sensitive and quantitative assays to measure light transmittance and focusing ability of human PS cell-derived micro-lenses have also been developed. This review will, therefore, examine how human PS cell-derived lens cells and micro-lenses might provide a new avenue for development of much-needed drugs to treat human cataract. [ABSTRACT FROM AUTHOR]