技术资料

ABSTRACTThe paradoxical exacerbation of cellular injury and death during reperfusion remains a problem in the treatment of myocardial infarction. Mitochondrial dysfunction plays a key role in the pathogenesis of myocardial ischemia and reperfusion injury. Dysfunctional mitochondria can be removed by mitophagy,culminating in their degradation within acidic lysosomes. Mitophagy is pivotal in maintaining cardiac homeostasis and emerges as a potential therapeutic target. Here,we employed beating human engineered heart tissue (EHT) to assess mitochondrial dysfunction and mitophagy during ischemia and reperfusion simulation. Our data indicate adverse ultrastructural changes in mitochondrial morphology and impairment of mitochondrial respiration. Furthermore,our pH-sensitive mitophagy reporter EHTs,generated by a CRISPR/Cas9 endogenous knock-in strategy,revealed induced mitophagy flux in EHTs after ischemia and reperfusion simulation. The induced flux required the activity of the protein kinase ULK1,a member of the core autophagy machinery. Our results demonstrate the applicability of the reporter EHTs for mitophagy assessment in a clinically relevant setting. Deciphering mitophagy in the human heart will facilitate development of novel therapeutic strategies. Summary: Mitochondrial dysfunction and lysosomal degradation of mitochondria (mitophagy) is induced after ischemia and reperfusion simulation in human engineered heart tissue,as shown with an endogenous pH-sensitive mitophagy reporter. View Publication

Acute liver failure (ALF) is a hepatology emergency with rapid hepatic destruction,multiple organ failures,and high mortality. Despite decades of research,established ALF has minimal therapeutic options. Here,we report that the small bioactive compound SCM-198 increases the survival of male ALF mice to 100%,even administered 24?hours after ALF establishment. We identify adiponectin receptor 2 (AdipoR2) as a selective target of SCM-198,with the AdipoR2 R335 residue being critical for the binding and signaling of SCM-198-AdipoR2 and AdipoR2 Y274 residue serving as a molecular switch for Ca2+ influx. SCM-198-AdipoR2 binding causes Ca2+ influx and elevates the phosphorylation levels of CaMKII and NOS3 in the AdipoR2-CaM-CaMKII-NOS3 complex identified in this study,rapidly inducing nitric oxide production for liver protection in murine ALF. SCM-198 also protects human ESC-derived liver organoids from APAP/TAA injuries. Thus,selectively targeting the AdipoR2-CaM-CaMKII-NOS3 axis by SCM-198 is a rapid-acting therapeutic strategy for advanced ALF. Late-stage acute liver failure (ALF) has limited therapies. The authors show that the bioactive compound SCM-198 extends the ALF treatment window from 3 to 24?hours in mice by selectively targeting the identified AdipoR2-CaM-CaMKII-NOS3-NO axis. View Publication

ABSTRACTObjectivesAutism spectrum disorder (ASD) is a neurodevelopmental condition that affects social communication and behaviors. While previous studies using animal models have substantially expanded our knowledge about ASD,the lack of an appropriate human model system that accurately recapitulates the human?specific pathophysiology of ASD hinders the precise understanding of its etiology and the development of effective therapies. This study aims to replicate pathological phenotypes in cerebral organoids derived from idiopathic ASD patients and to conduct proof?of?concept research for the development of ASD therapeutics.MethodsWe conducted an in vitro disease modeling study using cerebral organoids derived from three idiopathic ASD patients. Additionally,we performed organoid?based phenotypic drug screening to identify potential therapeutic compounds that could ameliorate the phenotypes observed in cerebral organoids derived from idiopathic ASD patients.ResultsHere we show that cerebral organoids derived from idiopathic ASD patients display malformation of the ventricular zones and impaired early neuronal differentiation. Through organoid?based phenotypic drug screening,we successfully generated cerebral organoids with normal tissue architecture in which the delayed neuronal differentiation could also be accelerated. Notably,cerebral organoids from ASD patients exhibited a reduced number of GABAergic neurons compared to healthy controls,resulting in an imbalance in the excitatory and inhibitory neuron ratio. The differentiation defects into GABAergic neurons in patient?derived cerebral organoids could be rescued by treating with either IGF1 or Gabapentin,a GABA agonist.ConclusionsOur findings provide a framework for utilizing patient?derived cerebral organoids in the development of personalized pharmaceutical treatment for ASD. Summary of in vitro disease modeling and drug screening using ASD patient?derived COs. This figure highlights the major phenotypes observed in COASD and the therapeutic effects of each compound screened in this study. View Publication

AbstractAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants,including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons,which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS,and TDP-43 modulates several mitochondrial transcripts. In this study,we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons,linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected,which correlated with downregulation of the motor protein complex,DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport,which can be partially rescued by DCTN1 overexpression. Dafinca et al. show that mutations in TDP-43 lead to decreased mitochondrial oxidative phosphorylation,partially due to interactions with the ATP production machinery and COX5A. These have direct effects on axonal transport,which is reduced in amyotrophic lateral sclerosis motor neurons,and overexpression of dynactin-1 significantly increases retrograde mitochondrial dynamics. Graphical Abstract Graphical Abstract View Publication

High-throughput massively parallel reporter assays (MPRAs) and phenotype-rich in vivo transgenic mouse assays are two potentially complementary ways to study the impact of noncoding variants associated with psychiatric diseases. Here,we investigate the utility of combining these assays. Specifically,we carry out an MPRA in induced human neurons on over 50,000 sequences derived from fetal neuronal ATAC-seq datasets and enhancers validated in mouse assays. We also test the impact of over 20,000 variants,including synthetic mutations and 167 common variants associated with psychiatric disorders. We find a strong and specific correlation between MPRA and mouse neuronal enhancer activity. Four out of five tested variants with significant MPRA effects affected neuronal enhancer activity in mouse embryos. Mouse assays also reveal pleiotropic variant effects that could not be observed in MPRA. Our work provides a catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays. MPRAs and in vivo transgenic mouse assays are two potentially complementary ways to assay the impact of noncoding variants. Here,authors find a strong and specific correlation between the assays in neural cells. Mouse assays also reveal pleiotropic effects not observed in MPRA. View Publication

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B,each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this,single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus,GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation. Using patient-derived induced pluripotent stem cells,the authors show that the transcription factor GFI1B and the lysine demethylase KDM1A/LSD1 promote gene programs while repressing those involved in myeloid differentiation. View Publication

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors,CoREST1,CoREST2,or CoREST3 (encoded by RCOR1,RCOR2,and RCOR3,respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST,in various neural and glial cell types during different developmental stages. However,there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap,we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons,as well as mature functional NGN2-induced neurons. Additionally,a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary,our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically,RCOR3 expression increases in neurons,while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans. Graphical abstractImage 1 Highlights•REST and RCOR genes display cell-type specific expression patterns in neural cells.•RCOR3 (encodes CoREST3) is upregulated during neuronal and astrocyte differentiation.•RCOR2 (encodes CoREST2) is downregulated during differentiation of astrocytes.•Evidence of potential cell-type specific functions of the CoREST family. View Publication

Simple SummaryAtypical teratoid rhabdoid tumors (ATRTs) and diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors that can resist chemotherapy and be influenced by their microenvironment. Astrocytes are the key components of the brain tumor microenvironment and can support tumor growth. We investigated the effects of astrocytes on cisplatin sensitivity in pediatric brain cancer cells. The crosstalk between astrocytes and cancer cells activated astrocytes and promoted cancer cell proliferation. Moreover,the tumor cells expressed elevated levels of drug resistance genes in the presence of astrocytes. In conclusion,astrocytes can significantly improve the growth of these tumor cells and modulate their chemosensitivity,highlighting their role in therapeutic resistance. AbstractBackground: ATRTs and DIPGs are deadly pediatric brain tumors with poor prognosis. These tumors can develop resistance to chemotherapies,which may be significantly influenced by their microenvironment. Since astrocytes are the most abundant glial cell type in the brain microenvironment and may support tumor growth and chemoresistance,this study investigated the effects of induced pluripotent stem cell-derived astrocytes (iPSC-astrocytes) on cisplatin sensitivity in CHLA-05-ATRT and SF8628 (DIPG) cells. iPSCs provide an unlimited and standardized source of nascent astrocytes,which enables modeling the interaction between childhood brain tumor cells and iPSC-astrocytes within a controlled coculture system. Methods: To study the effects on tumor growth,the iPSC-astrocytes were cocultured with tumor cells. Additionally,the tumor cells were exposed to various concentrations of cisplatin to evaluate their chemosensitivity in the presence of astrocytes. Results: The paracrine interaction of iPSC-astrocytes with tumor cells upregulated astrocyte activation markers GFAP and STAT3 and promoted tumor cell proliferation. Moreover,the cisplatin treatment significantly decreased the viability of CHLA-05-ATRT and SF8628 cells. However,tumor cells exhibited reduced sensitivity to cisplatin in the coculture with iPSC-astrocytes. During cisplatin treatment,DIPG cells in particular showed upregulation of resistance markers,ERK1,STAT3,and MTDH,which are associated with enhanced proliferation and invasion. They also had increased expression of APEX1,which is involved in the base excision repair pathway following cisplatin-induced DNA damage. Conclusion: These findings underscore the significance of the tumor microenvironment in modulating tumor cell survival and chemosensitivity. View Publication

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types,making further research difficult. Here,we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology,doubling rate,ability to form colonies,surface marker analysis,and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology,growth kinetics,and surface marker characterization. In contrast,the serum-containing medium gives better potential for further MSC differentiation into osteogenic,chondrogenic,and adipogenic lineages. View Publication

Biallelic mutations in PIGK cause GPI biosynthesis defect 22 (GPIBD22),characterized with developmental delay,hypotonia,and cerebellar atrophy. The understanding of the underlying causes is limited due to the lack of suitable disease models. To address this gap,we generated a mouse model with PIGK deficits,specifically in Purkinje cells (Pcp2-cko) and an induced pluripotent stem cell (iPSC) model using the c.87dupT mutant (KI) found in GPIBD22 patients. Pcp2-cko mice demonstrated cerebellar atrophy,ataxia and progressive Purkinje cells loss which were accompanied by increased apoptosis and neuroinflammation. Similarly,KI iPSCs exhibited increased apoptosis and accelerated neural rosette formation,indicating that PIGK defects could impact early neural differentiation that confirmed by the RNA-Seq results of neural progenitor cells (NPCs). The increased apoptosis and accelerated NPC differentiation in KI iPSCs are associated with excessive unfolded protein response (UPR) pathway activation,and can be rescued by UPR pathway inhibitor. Our study reveals potential pathogenic mechanism of GPIBD22 and providing new insights into the therapeutic strategy for GPIBD. View Publication

ABSTRACTHuman induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However,their differentiation into specific cell lineages,such as definitive endoderm (DE) and pancreatic progenitor (PP),often suffers from poor reproducibility,due partially to their pluripotency. In this work,we investigated the impact of iPSC confluency during cell self?renewal and seeding density on cell metabolic activity,glycolysis to oxidative phosphorylation shift,and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual?positive cells in PPs. Interestingly,we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. Graphical Abstract and Lay SummaryHuman induced pluripotent stem cell (iPSC) differentiation into specific cell lineages often shows poor reproducibility due to cell pluripotency. This study demonstrated impact of iPSC seeding strategy on metabolic activity and differentiation potential,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. View Publication

Stargardt disease (STGD),predominantly caused by mutations in the ABCA4 gene,is a leading cause of inherited retinal degeneration. Although several lines of mice expressing disease-causing variants have been produced,mice due to the lack of macular may not be the perfect model to mimic the characteristics of STGD. To address this knowledge gap,we generated retinal organoids from patient-derived induced pluripotent stem cells (iPSCs) harboring ABCA4 mutations and performed biological validation. The generated retinal organoids were subjected to single-cell RNA sequencing (scRNA-seq) at major developmental stages (40,90,150,200,and 260 days),and we additionally compared the transcriptomics with our recently published control retinal organoids to further confirm the reliability of the dataset. By using iPSCs carrying most common variant in Chinese STGD patients,the dataset not only provides a powerful resource for studying STGD,but also offers novels insight into the developmental mechanisms underlying ABCA4-associated pathological changes in the retinal organoid system. View Publication