Abstract Otic neurons, also known as spiral ganglion neurons (SGNs) in mammalian cochlea, transmit electrical signals from sensory hair cells to cochlear nuclei of the auditory system. SGNs are sensitive to toxic insults, vulnerable to get irreversible damaged and hardly regenerate after damage, causing persistent sensorineural hearing loss. Yet, to get authentic SGNs for research or therapeutic purpose remains challenging. Here we developed a protocol to generate human otic neuronal organoids (hONOs) from human pluripotent stem cells (hESCs), in which hESCs were step‐wisely induced to SGNs of the corresponding stages according to their developmental trajectory. The hONOs were enriched for SGN‐like cells at early stage, and for both neurons and astrocytes, Schwann cells or supporting cells thereafter. In these hONOs, we also determined the existence of typical Type I and Type II SGNs. Mature hONOs (at differentiation Day 60) formed neural network, featured by giant depolarizing potential (GDP)‐like events and rosette‐organized regions‐elicited calcium traces. Electrophysiological analysis confirmed the existence of glutamate‐responsive neurons in these hONOs. The otic neuronal organoids generated in this study provide an ideal model to study SGNs and related disorders, facilitating therapeutic development for sensorineural hearing loss.
To the Editor, Acute lung injury (ALI) and the inflammatory cytokine storm cause considerable amount of deaths in the COVID-19 pandemic.1, 2 Currently, very limited therapeutic options are available for the COVID-19-induced ALI. In our preclinical experiments,3 we found that a single intravenous transfusion of immunity- and matrix-regulatory cells (IMRCs), derived from fully differentiated human embryonic stem cells, could safely treat ALI by rapidly modulating the inflammation induced by pulmonary cell death.3 Encouraged by this result, as part of an expanded access programme, we pilot-tested GMP-grade IMRC transfusion as a compassionate treatment in a severely ill COVID-19 patient who was diagnosed with ALI. A 44-year-old male patient from Wuhan was admitted on 23 January 2020, presenting with a 6-day history of fever and cough. The physical examination revealed a fever of 37.9°C, blood pressure of 120/61 mm Hg, pulse rate of 80 beats per minute, respiratory rate of 21 breaths per minute and blood oxygen saturation of 97.9%. Laboratory testing showed lymphocytopenia with a lymphocyte count of 0.65 × 109 cells per litre. Nasopharyngeal swab specimen was collected and tested positive for SARS-CoV-2 by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). He was briefly treated with Lianhua Qingwen (herbal flu drug) and the anti-retroviral cocktail lopinavir/ritonavir (Kaletra), but showed no improvement. By January 28, the fever had risen to 39°C, and he had shortness of breath under oxygen supplementation. CT scans showing multiple ground-glass opacities indicated the pneumonia had progressed to a severe stage (Figure 1A). On January 30, his resting blood oxygen saturation fell to 91%, and he was diagnosed as a severely ill patient with ALI. After the patient's consent, he was intravenously infused with 3 × 106 IMRCs per kilogram body weight the same day. He was given a second infusion of IMRCs with the same dose mentioned above with interval seven days. The patient's blood pressure rose from 127/76 mm Hg to 158/106 mm Hg after the first IMRC infusion, and he was given 10 mg nifedipine orally on infusion days 1, 4, 5 and 6. The patient showed no signs of discomfort except for transient high blood pressure. By February 2, the patient no longer had shortness of breath, and his resting blood oxygen saturation rose to 95%. By February 5 (first infusion day 7), the patient's throat swab tested negative for SARS-CoV-2 by qRT-PCR, which was reconfirmed on February 7. However, chest CT scans showed that the pneumonia was still in the advanced stage. On hospital day 15 (illness day 21, first infusion day 8), the patient still had lymphocytopenia with a lymphocyte count of 0.79 × 109 cells per litre. On hospital day 17 (illness day 23, first infusion day 10), the patient recovered from lymphocytopenia with a lymphocyte count of 1.02 × 109 cells per litre (Table S1). On hospital day 20 (illness day 26, first infusion day 13), after showing significant recovery by CT scans, the patient was recommended for discharge. On March 2 (first infusion day 33), one month after the first IMRC infusion, follow-up CT scans showed that the patient had completely recovered. Cytokines in the patient's plasma were measured on first infusion day 1, first infusion day 8 (second infusion day 1) and first infusion day 11 (second infusion day 4) (Table S2). Most pro-inflammatory cytokines showed a marked decrease, including G-CSF, M-CSF, GM-CSF, IL-1α, IL-1β, IL-5, IL-6, IL-7, IL-8, IL-15 IL-17E/IL-25, IL-27, IP10/CXCL10, MCP-1/CCL2, MCP-2/CCL7, MIG/CXCL9, MIP-1α/CCL3 and GRO-α/CXCL1 (Figure 1B). There was also an increase in immunomodulatory cytokines, such as IL-1RA, IL-12, IL-22 and MDC/CCL22 (Figure 1C). To the best of our knowledge, this is the first time that pure hESC-derived IMRCs have been intravenously infused into the human body. No severe adverse events were observed one month after IMRC infusion. Further work will determine whether this therapy can be replicated widely. This work was supported by National Emergency Project of the Ministry of Science and Technology of China (2020YFC0843900 and 2020YFC0841900), Beijing Municipal Science & Technology Commission (Z181100003818005), National Key Research and Development Program (2016YFA0101502), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030701, XDA16040502), and International Partnership Program of Chinese Academy of Sciences (152111KYSB20160004). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. BH, RJ and JH conceived the project. ZL, TG and BG, provided the testing results of IMRCs. WH, FJ, HD and WL analysed clinical case. JW, ZH, LW and Y.T designed the research and wrote the manuscript with help from all of the authors. Drs. JW, ZH, LW and YT contributed equally to this case report. B. H., JH and R. J. are the corresponding authors of this case report. All authors read and approved the final manuscript. The study is approved by the Ethics Committee of Beijing Youan Hospital, Capital Medical University, Beijing, China. ClinicalTrials.gov Identifier: NCT04331613. https://www.clinicaltrials.gov/ct2/show/NCT04331613?term=Castem&draw=2&rank=1 The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Human embryonic stem cells (hESCs) play an important role in regenerative medicine due to their potential to differentiate into various functional cells. However, the conventional adherent culture system poses challenges to mass production of high-quality hESCs. Though scientists have made many attempts to establish a robust and economical hESC suspension culture system, there are existing limitations, including suboptimal passage methods and shear force caused by dynamic stirring. Here, we report on an efficient large-scale culture system, which enables long-term, GMP grade, single-cell inoculation, and serial expansion of hESCs with a yield of about 1.5 × 109 cells per 1.5-L culture, while maintaining good pluripotency. The suspension culture system was enlarged gradually from a 100-mm dish to a 1.8-L culture bag with methylcellulose involvement to avoid sphere fusion. Under the optimal experimental protocol, this 3D system resolves current problems that limit mass production and clinical application of hESCs, and thus can be used in commercial-level hESC production for cell therapy and pharmaceutics screening in the future.
Abstract 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.
Midbrain dopaminergic (DA) progenitors derived from human pluripotent stem cells are considered to be a promising treatment for Parkinson's disease (PD). However, the differentiation process produces undesired cell types, which influence the in vivo evaluation of DA cells. In this paper, we analyze the cell fate choice during differentiation and provide valuable information on cell preparation.Human embryonic stem cells were differentiated into DA progenitors. We applied single-cell RNA sequencing (scRNA-seq) of the differentiation cells at different time points and investigated the gene expression profiles. Based on the differentially expressed genes between DA and non-DA cells, we investigated the impact of LGI1 (DA enriched) overexpression on DA differentiation and the enrichment effect of CD99 (non-DA enriched) sorting.Transcriptome analyses revealed the DA differentiation trajectory as well as non-DA populations and three key lineage branch points. Using genetic gain- and loss-of-function approaches, we found that overexpression of LGI1, which is specific to EN1+ early DA progenitors, can promote the generation of TH+ neurons. We also found that choroid plexus epithelial cells and DA progenitors are major components of the final product (day 25), and CD99 was a specific surface marker of choroid plexus epithelial cells. Sorting of CD99- cells eliminated major contaminant cells and improved the purity of DA progenitors.Our study provides the single-cell transcriptional landscape of in vitro DA differentiation, which can guide future improvements in DA preparation and quality control for PD cell therapy.
The interactions between extra-embryonic tissues and embryonic tissues are crucial to ensure proper early embryo development. However, the understanding of the crosstalk between the embryonic tissues and extra-embryonic tissues is lacking, mainly due to ethical restrictions, difficulties in obtaining natural human embryos, and lack of appropriate in vitro models. Here by aggregating human embryonic stem cells (hESCs) with human trophoblast stem cells (hTSCs), we revealed the hESCs robustly self-organized into a unique asymmetric structure which the primitive streak (PS) like cells exclusively distributed at the distal end to the TS-compartment, and morphologically flattened cells, presumed to be the extra-embryonic mesoderm cells (EXMC) like cells, were induced at the proximal end to hTSCs. Our study revealed two potential roles of extra-embryonic trophectoderm in regulating the proper PS formation during gastrulation and EXMCs induction from the human epiblast.
Synucleinopathies such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are characteristic for α -synuclein aggregates in neurons or glia, and are always manifested olfaction deficits at their primary onsets. It remains elusive why aggregation of α -synuclein predominantly affect the olfactory system. Employing the knockout mice, we investigate the physiological function of α-synuclein in olfactory system. We found that deletion of α-synuclein primarily interferes the projection of olfactory sensory neurons. iTRAQ based LC-MS identified that 188 proteins are differentially expressed, including 9 that were associated with axon guidance. Among them, NCK2 is most significantly down-regulated, which was indicated to be involved a PPI network of 21 proteins, including 11 players of the Ephrin receptor signaling pathway. Either α-synuclein deletion or NCK2 deficiency can inactivate Eph A4 receptor. Re-expressing α-synuclein in the α-synuclein knockout neurons reverse the NCK2, as well as the phosphorylated Eph A4 (the activated Eph A4). Thus, α-synuclein regulates axon guidance through NCK2-Eph A4 signaling pathway. Malfunction of α-synuclein, whether because of deletion or aggregation, may cause aberrant olfactory neurons projection and subsequent olfaction deficits. This extended our knowledge of effects of α-synuclein in olfactory system, which may explain why olfaction is usually impaired in some synucleinopathy related disorders such as Parkinson’s disease.
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