Synthetic Circuits Reveal the Key to Rewinding the Cellular Clock – The Scientist

Most people wonder how their lives would change if they could turn back time and remake past decisions. While a seemingly impossible feat, stem cell biologists Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University first turned back the cellular clock in 2006.1 By overexpressing four transcription factors in fully differentiated fibroblasts, Takahashi and Yamanaka reprogrammed the cells to a pluripotent state and called them induced pluripotent stem cells (iPSC).

Although researchers employ iPSC in the laboratory and the clinic, scientists struggle to efficiently produce large quantities of iPSC.2 Reprogramming is still very inefficient, said Thorsten Schlaeger, a stem cell biologist at the Boston Childrens Hospital. It is still not fully understood why 98 or 99.9 percent of the cells do not end up reprogramming into iPSC. In a recently published Science Advances paper, Schlaeger and his team developed a system for tracking the fate of cells with different transcription factor expression dynamics during reprogramming.3 These findings will enable researchers in the field to improve iPSC yield.

Scientists suspected that the heterogeneity in reprogramming outcomes results from variations in the levels and durations of transcription factors expression. Consequently, several research groups have attempted to correlate the success of reprogramming to the levels of the octamer-binding transcription factor 4 (OCT4), which is one of Takahashi and Yamanakas transcription factors that is essential for the reprogramming process. However, these studies used population-based measurements and failed to consider the contribution of endogenous OCT4 to reprogramming.

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Compelled to overcome these limitations, Schlaegers coauthor Domitilla Del Vecchio, a discipline-straddling mechanical engineer at the Massachusetts Institute of Technology, developed an innovative OCT4 expression system. The idea really was to try to use a more sophisticated way of overexpressing transcription factors to reprogram stem cells, Del Vecchio recalled.

Del Vecchios team designed a synthetic gene circuit to ectopically overexpress a fluorescently-tagged version of the OCT4 transcription factor, while simultaneously blocking the expression of the endogenous OCT4 through microRNA. This allowed the researchers to control the total OCT4 protein levels within the cell and quantify them by measuring the fluorescence. Additionally, the ectopic OCT4 gene was controlled by an inducible and noisy promoter, which meant that the system generated variability in the expression of the OCT4 conjugate and a broad range of trajectories to assess, such as cells that maintained high OCT4 expression throughout reprogramming or those whose expression decreased over time.

Thorsten Schlaeger and Domitilla Del Vecchio developed a synthetic gene circuit-based system that allowed them to control and monitor the total OCT4 protein levels within fibroblasts during reprogramming.

Thorsten Schlaeger and Domitilla Del Vecchio

To determine which OCT4 trajectories successfully reprogrammed human dermal fibroblasts into iPSC, Del Vecchio, Schlaeger, and their team transduced the cells with lentiviral vectors encoding their OCT4 trajectory generator and followed the levels of fluorescently-tagged OCT4 proteins within the cells over time through imaging. The researchers then fixed the resulting cell colonies and immunostained them for two pluripotent stem cell surface markers.

They observed that the colonies fell into three categories: type I colonies were positive for only one of the surface markers; type II colonies showed the exact opposite staining pattern from type I; and type III colonies were positive for both markers. They considered cells within type III colonies as iPSC and categorized the cells within type I and II colonies as incompletely reprogrammed. Despite these differences, cells in all three colony types stably expressed supraphysiological levels of OCT4 during reprogramming, indicating that successfully reprogramming human dermal fibroblasts into iPSC requires consistently high levels of the OCT4 transcription factor. But this parameter alone is not sufficient to promote iPSC generation.

The paper is innovative in a technical sense. It is consistent with work that has been done showing that elevated levels of OCT4 are important for the reprogramming process, said Dean Tantin, a geneticist at the University of Utah, who was not involved in the study.

Although Tantin thought Del Vecchios system presented a clever way to directly examine total OCT4 protein levels within live cells, he suggested that examining protein levels alone may not convey the whole story. The level of a protein based on a fluorescent marker is not the same as the activity of a proteinits ability to bind DNA [or] its ability to regulate transcription once it binds," he noted. "So, I think where the field needs to go now is [to find out] how OCT4 activity is really dynamically regulated during [reprogramming]. he noted. Building on this idea, Tantin and his team recently determined that OCT4 activity during reprogramming and differentiation is redox-regulated, and he suspects that regulation of other reprogramming components will be of interest in the years to come.4

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Del Vecchio hopes that her work will inspire other researchers to think beyond the standard methods they employ to dissect molecular pathways. This study is showing how more sophisticated genetic engineering tools can be used in the context of a highly complex biological process and help you get information that will be difficult to get otherwise, Del Vecchio said.

Schlaeger wants to leverage the knowledge gained in this study to develop off-the-shelf iPSC-based therapies, such as CAR T cells, and he believes that precision engineering will be the key to safely bringing these products to the clinic. With the cells, we want to get to this precise control and that can only be done with complex genetic switches and circuits, Schlaeger said.

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Synthetic Circuits Reveal the Key to Rewinding the Cellular Clock - The Scientist

PLEKHM2 deficiency induces impaired mitochondrial clearance and elevated ROS levels in human iPSC-derived … – Nature.com

Generation of homozygous PLEKHM2-KO hiPSCs and differentiating into cardiomyocytes

The generation of homozygous PLEKHM2-KO hiPSCs was carried out using the CRISPR-Cas9 system, and the guide RNA (gRNA) was designed and synthesized to target exon 2 of PLEKHM2 (Fig. 1A). Subsequent screening confirmed the successful knockout of PLEKHM2 gene, which revealed a one-nucleotide deletion in one allele and a one-nucleotide insertion in another allele (Fig. 1B and Supplementary Fig. 1B). The cell line under investigation was found to express the human pluripotency markers SSEA4 and OCT4 (Fig. 1C) and tested negative for mycoplasma contamination (Supplementary Fig. 1C). Western blot (WB) analyses revealed the absence of PLEKHM2 protein expression in PLEKHM2-KO hiPSC-CMs at day 20 (Fig. 1D). Wild-type (WT) and PLEKHM2-KO hiPSCs were differentiated into cardiomyocytes using the small molecule-based method (Fig. 1E). The efficacy of hiPSC-CMs differentiation was evaluated using flow cytometry, which indicated that PLEKHM2-KO and WT hiPSC-CMs exhibited similar proportions of cardiac Troponin T (cTnT)-positive cells (around 93%) at day 20 post differentiation (Fig. 1F, G). These results indicate that the PLEKHM2-KO hiPSC-CMs were successfully constructed.

A The PLEKHM2 gene structure and the location of the guide RNA (gRNA) used for epigenome editing with CRISPR/Cas9. B Sequencing analysis confirmed a homozygous PLEKHM2-KO hiPSC line with a 1-nucleotide deletion in one allele and a 1-nucleotide insertion in the other allele. C Pluripotent stem cell markers SSEA4 and OCT4 were detected by immunofluorescence staining in PLEKHM2-KO colonies. Scale bar, 20 m. D Western blot analysis of PLEKHM2 in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at day 20. E Protocol of small molecule-based methods to induce cardiac differentiation. F, G Flow cytometry analysis for cTnT from representative WT and PLEKHM2-KO differentiation at day 20. The results are presented as meansSD of 3 independent experiments. N.S. not significant.

We next investigate the dynamic changes in myocardial contractility and calcium transients of PLEKHM2-KO hiPSC-CMs. The HCell series single myocardial cell function detection system was utilized to measure myocardial contractility [10] (Supplementary Fig. 2A), and the green fluorescent calcium-modulated protein 6 fast type (GCaMP6f) calcium imaging system was employed to track myocardial calcium transients [11] (Supplementary Fig. 2B, C).

At the early stage of myocardial differentiation, specifically on 20 day, no significant alterations in myocardial contractility were observed between the WT hiPSC-CMs and the PLEKHM2-KO hiPSC-CMs. But at day 30, PLEKHM2-KO hiPSC-CMs exhibited a minor reduction in systolic displacement, as well as systolic and diastolic velocities compared to WT hiPSC-CMs, but no change in contractile force. And at day 40, the systolic displacement, contractile force, as well as systolic and diastolic velocities were significantly reduced in PLEKHM2-KO hiPSC-CMs compared to the WT hiPSC-CMs (Fig. 2AE, and Supplementary Fig. 2D, E), showing that the PLEKHM2-KO hiPSC-CMs developed systolic dysfunction phenotype. Calcium transient is a principal mechanism responsible for myocardial contraction, wherein the magnitude of contraction force is contingent upon variations in calcium ion concentration within the cell [12]. Hence, we next evaluated the alterations in calcium transients in the myocardium. In accordance with the trend observed in myocardial contractility, no significant variation was detected in calcium transient of PLEKHM2-KO hiPSC-CMs during the early phase post myocardial differentiation. However, a decline in calcium transient amplitude was observed at day 30, alongside a decrease in upstroke and recovery velocity of calcium transients in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs, and further exacerbated by 40 day (Fig. 2FJ, and Supplementary Fig. 2F). Interestingly, we also found that compared to WT hiPSC-CMs, the baseline values of calcium transients in PLEKHM2-KO hiPSC-CMs showed a significantly increased at day 40, indicating abnormal calcium handling in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 2G). These results suggest that abnormal calcium handling is a potential cause of the impaired myocardial contractility in PLEKHM2-deficient cardiomyopathy.

A Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 20, 30, and 40. BE Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs (n=12 cells per group). F Representative line scan images of calcium transients in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 20, 30, and 40. GJ Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs (n=12 cells per group). K Quantitative PCR analysis of heart failure and calcium handling -related genes in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 40. Data are shown as meanSD of 3 independent experiments. L Representative immunofluorescence staining and transmission electron microscope (TEM) of sarcomeric. M Quantification of complete organization, intermediate disorganization, and complete organization in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 40 based to immunofluorescence staining (more than 120 cells per group). Scale bar, 10 m. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S. not significant.

Subsequently, we evaluated the expression of key genes involved in heart failure and calcium handling. We found a significant increase in the expression of both NPPB and the MYH7/MYH6 ratio in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs, whereas ryanodine receptor 2 (RYR2) expression significantly decreased at day 40 (Fig. 2K). Moreover, we observed significantly disordered sarcomeres in PLEKHM2-KO hiPSC-CMs at day 40 (Fig. 2L, M). Moreover, transmission electron microscopy (TEM) showed significant abnormalities in the myofilaments of PLEKHM2-KO hiPSC CMs, including disordered myofilament arrangement and blurred Z-disc morphology (Fig. 2L). Overall, these findings corroborate the strong link between PLEKHM2 deficiency and DCM, which manifests as reduced contractility and impaired calcium handling, along with sarcomeric disorganization and dysregulated expression of heart failure markers.

To assess for potentially pathogenic effects of PLEKHM2-deficient cardiomyopathy, we performed quantitative transcriptome profiling by RNA-seq (Supplementary Fig. 3AC). We identified 8725 differentially expressed genes in PLEKHM2-KO hiPSC-CMs versus WT hiPSC-CMs at day 40, including 4426 upregulated and 4299 downregulated genes (Fig. 3A). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that these dysregulated genes were enriched in pathways mainly involved in regulating autophagy, lysosome, cardiomyopathy, apoptosis and metabolism (Fig. 3B). Of particular a significant finding was that the dysregulated genes were enriched in autophagy with the highest enrichment score in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs (Fig. 3B). The molecular-level analysis of Gene Ontology (GO) enrichment demonstrated a marked dysregulation in gene expression related to mitochondria, apoptosis, and autophagy in PLEKHM2-KO hiPSC-CMs (Fig. 3C, D). Notably, mitochondria-related pathways show the most significant differences between PLEKHM2-KO and WT hiPSC-CMs (Fig. 3C). Overall, these results indicate that PLEKHM2 deficiency leads to widespread dysregulation of signaling pathways in cardiomyocytes. Subsequently, we conducted quantitative PCR to validate the expression of genes associated with substantial dysregulation of mitochondria, apoptosis, and autophagy in RNA seq. Our results indicate a significant downregulation of BNIP3, DNM1L, OPA1, and MFN1 in addition to an upregulation of TSPO expression in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs at day 40 (Fig. 3E). It is widely acknowledged that BNIP3 and TSPO participate in various physiological processes such as mitophagy, apoptosis, oxidative stress, and the oxidative respiratory chain [13, 14]. While DNM1L, OPA1, and MFN1 play crucial roles in maintaining and regulating mitochondrial morphology and stability [15, 16]. The observed dysregulation in these genes highlight a disruption of mitochondrial homeostasis in PLEKHM2-deficient cardiomyopathy.

A Volcano plot shows 8725 genes with altered expression in PLEKHM2-KO hiPSC-CMs compared with WT. Blue and red dots indicate genes with increased and decreased expression, respectively, based on a P value<0.05 and a log2 fold change >1 (n=3 for each group). B Enrichment analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) databases revealed that pathways related to lysosomal function, autophagy, cardiomyopathy, apoptosis and metabolism were disrupted in PLEKHM2-KO hiPSC-CMs. ARVC, arrhythmogenic right ventricular cardiomyopathy. C Gene Ontology (GO) enrichment analysis showed significant changes in gene expression associated with mitochondrial function, apoptosis and autophagy in PLEKHM2-KO hiPSC-CMs. The color scale indicates the P values of the top 15 altered pathways in GO molecular function and the bubble size reflects the number of genes involved in each pathway. D Heatmap of differentially expressed genes involved in mitochondrial function, apoptosis and autophagy. E Quantitative PCR confirmed the altered expression of a representative subset of genes identified by RNA sequencing in PLEKHM2-KO hiPSC-CMs. Data are shown as meanSD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

RNA-seq analysis revealed notable anomalies in autophagy and mitochondrial-related pathways, as indicated by significant findings in the KEGG and GO enrichment analysis. Following this discovery, we proceeded to investigate alterations in mitochondrion and autophagic processes. To further investigate the impacts of PLEKHM2 deficiency on the mitochondrion, we next assessed mitochondrial morphology and content using Mitotracker at day 40. Compared to the typical linear arrangement of mitochondria along sarcomeres in WT hiPSC-CMs, the mitochondria within PLEKHM2-KO hiPSC-CMs display distinctive fragmented and punctate patterns, along with irregular distribution throughout the cytoplasm (Fig. 4A, and Supplementary Fig. 4A, B). TEM revealed matrix swelling, empty spaces, and loose, disordered, and wider cristae in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 4A, C). This suggest that the PLEKHM2 deficiency significantly affects the localization and tissue structure of mitochondria in cardiomyocytes. Mitochondrial morphology disruption usually trigger mitophagy, which targets damaged or dysfunctional mitochondria for degradation and clearance from the cell, and the number of mitochondria within the cell usually decrease due to their removal [17]. However, further analysis using flow cytometry revealed an increasing mitochondrial content within the PLEKHM2-KO hiPSC-CMs, compared to the WT hiPSC-CMs (Fig. 4B, C). These results suggested that mitochondrial morphological abnormalities and impaired mitochondrial clearance occur in PLEKHM2-KO hiPSC-CMs.

A Mitotracker staining revealed that PLEKHM2-KO altered the mitochondrial structure from the filamentous form aligned with the sarcomere in WT hiPSC-CMs to a punctate and fragmented morphology at day 40. Scale bar, 10 m. B, C Quantification of Mitotracker green intensity obtained by flow cytometry demonstrates a significantly increased fluorescence intensity in PLEKHM2-KO hiPSC-CMs at day 40 as compared with WT hiPSC-CMs (n=4). DF Autophagic flux was assessed in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs using mRFP-EGFP-LC3 adenovirus and subjected them to starvation medium for 0, 1, 2, and 4h at day 40. Representative images and quantification of GFP+, RFP+, and GFP, RFP+ puncta are shown in (D)(F). 12 cells per cell line per condition were analyzed. Scale bars, 10 m. G, H Representative western blot and quantification of P62 expression in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at day 40 (n=3). CQ: chloroquine. Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S. not significant.

The damaged mitochondria undergo degradation and digestion with the participation of lysosomes [4], while impaired autophagy or lysosomal acidification disorders usually impair mitophagy, resulting in delayed clearance of damaged mitochondria [18]. Consequently, we proceeded to investigate alterations in lysosomal localization and autophagy in the PLEKHM2-KO hiPSC-CMs. In this study, we utilized LAMP1 as a marker to identify lysosomal localization. Our results indicate that lysosomes in PLEKHM2-KO hiPSC-CMs exhibit significant clustering around the nucleus, whereas the lysosomes in the WT hiPSC-CMs demonstrate scattered distribution throughout the cytoplasm (Supplementary Fig. 4C, D), which is consistent with previous study [2]. To investigate the effects of PLEKHM2 deficiency on autophagy, we next monitored alterations in autophagic flux at 0, 1, 2, and 4hours post-starvation. Day 3 after Ad-mRFP-EGFP-LC3 infection, we observed a significant accumulation of autophagosomes (GFP+/RFP+ puncta) with increasing starvation duration in both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs. Notably, at the 2-hour after starvation, the number and proportion of autophagosome-lysosome fusion (GFP-/RFP+ puncta) in PLEKHM2-KO hiPSC-CMs was significantly lower than that of the WT hiPSC-CMs, indicating that the autophagic degradation of PLEKHM2-KO hiPSC-CMs was impaired (Fig. 4DF). In this study, we observed that in the late phase of autophagy (4hour after staving), most autophagosomes in the WT group accumulated around the nucleus and fused with lysosomes to form autolysosomes [19]. However, in the PLEKHM2-KO hiPSC-CMs, a substantial number of autophagosomes remained scattered within the cytoplasm and were not yet concentrated around the nucleus (Supplementary Fig. 4E), suggesting that PLEKHM2 deficiency affected the aggregation of autophagsomes to the perinuclear and fusion with lysosomes. In the subsequent WB results, we also observed that PLEKHM2 deficiency led to accumulation of p62 (Fig. 4G, H, and Supplementary Fig. 4F, G). In summary, these findings suggested that PLEKHM2 deficiency lead to abnormal lysosomal localization and blocking of autophagic flux, resulting in impaired autophagy and damaged mitochondrial accumulation.

Mitophagy is a fundamental cellular self-cleaning mechanism that plays a critical role in maintaining mitochondrialfunction and preventing the accumulation of reactive oxygen species (ROS) by selectively removing damaged mitochondria [20, 21]. m is a crucial indicator of mitochondrial health and function. To investigate the impact of PLEKHM2 deficiency on mitochondrial function, the carbocyanine compound JC-1, a fluorescent voltage-sensitive dye that possesses membrane-permeant fluorescent lipophilic cationic properties, was utilized to assess m and mitochondrial health. Our results revealed that JC-1 in PLEKHM2-KO hiPSC-CMs, exhibited a robust red fluorescence and weak green fluorescence similar to the WT hiPSC-CMs at day 20. However, over time, the red fluorescence of JC-1 in the PLEKHM2-KO hiPSC-CMs decreased gradually, while the green fluorescence increased (Fig. 5A). Notably, at day 30 and 40, the ratio of aggregate to monomeric JC-1 fluorescence in the PLEKHM2-KO hiPSC-CMs significantly reduced compared to that of the WT hiPSC-CMs (Fig. 5B). Futhrtmore, the destabilization in m lead to the release of cytC from mitochondria, which activated the caspase-3 in PLEKHM2-KO hiPSC-CMs at 40 day (Supplementary Fig. 5AC). These results suggest that PLEKHM2 deficiency leads to extensive depolarization of m and impaired mitochondrial function.

A, B Representative immunofluorescence staining and quantification of JC-1 revealed that mitochondrial monomers (green fluorescence) increased and the mitochondrial aggregates (red fluorescence) decreased gradually in PLEKHM2-KO hiPSC-CMs compared to WT at day 20, 30, and 40 (more than 120 cells per group). Scale bar, 10 m. C Heatmap of differentially expressed genes involved in oxidative stress in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs. D GSEA analysis revealed dysregulation of the respose to oxidative stress signaling pathway in PLEKHM2-KO hiPSC-CMs. E, F Representative flow cytometry analysis and quantification of cell reactive oxygen species (ROS) intensity demonstrated a continuous increased fluorescence intensity in PLEKHM2-KO hiPSC-CMs at days 20, 30, and 40 as compared with WT hiPSC-CMs. G, H Oxygen consumption rate (OCR) of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at 40 day was measured using a seahorse analyzer. Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

Numerous studies have shown that ROS plays a crucial role in inducing widespread m depolarization by directly triggering mPTP opening. And mPTP opening can further enhance ROS production by impairing m, ultimately triggering a vicious cycle of m depolarization and ROS production [22,23,24]. RNA-seq suggested significant changes in the expression gene profile associated with oxidative stress in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs (Fig. 5C, D). Thus, to investigate whether the PLEKHM2 deficiency leads to an increase in ROS levels, we assessed the dynamic changes in ROS levels in PLEKHM2-KO hiPSC-CMs. We found a continuous increase in ROS levels in PLEKHM2-KO hiPSC-CMs than WT hiPSC-CMs (Fig. 5E, F), which indicated that PLEKHM2 deficiency causes progressive oxidative stress in hiPSC-CMs. To further investigate the effect of PLEKHM2 deficiency on mitochondrial OXPHOS activity, the oxygen consumption rates (OCR) of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs was analyzed at day 40 (Fig. 5G). These kinetic results revealed that PLEKHM2 deficiency significantly impaired ATP production, basal respiration and spare capacity (Fig. 5H). These results suggest that PLEKHM2 deficiency causes extensive mitochondrial dysfunction.

Previous studies have shown a strong link between oxidative stress and cardiomyopathy. To investigate whether ROS plays an important role in the pathogenesis of PLEKHM2-deficient cardiomyopathy, we administered oxidative stress activator, lipopolysaccharides (LPS) to WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs, and observed the effects on myocardial mitochondrial function, calcium handling, and contractility at day 40. After LPS administration, both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs exhibited significantly higher levels of ROS than untreated CMs (Fig. 6A, B). Next, the JC-1 was utilized to assess the effect of LPS treatment on mitochondrial function. Our results showed that LPS treatment induced the same mitochondrial dysfunction phenotype in WT hiPSC-CMs as in PLEKHM2-KO hiPSC-CMs. Moreover, LPS treatment exacerbated the m destabilization in PLEKHM2-KO hiPSC-CMs compared to untreated hiPSC-CMs (Fig. 6C, D). To investigate whether oxidative stress accelerates the progression of PLEKHM2-deficient cardiomyopathy, we evaluated the effects of LPS administration on the calcium transient and myocardial contractility of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs. We found that LPS treatment decreased calcium transients (Fig. 6EI, and Supplementary Fig. 6) in both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs compared to untreated hiPSC-CMs. And LPS treatment significantly decreased myocardial contractility (Fig. 6JN) in WT hiPSC-CMs compared to untreated hiPSC-CMs. These results suggested that oxidative stress may play a significant role in mitochondrial dysfunction, abnormal calcium handling and impaired myocardial contractility in the development of PLEKHM2-deficient cardiomyopathy.

A, B Representative flow cytometry analysis and quantification of cellular ROS levels showed that both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs had significantly increased ROS production compared to untreated controls after LPS exposure (n=4 independent experiments). C, D Representative immunofluorescence staining and quantification of JC-1 revealed that LPS treatment impaired mitochondrial membrane potential of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs, as indicated by increased green fluorescence (monomeric form) and decreased red fluorescence (aggregated form) of JC-1 (more than 120 cells per group). Scale bar, 10 m. E Representative line scan images of calcium transients in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without LPS treatment at day 40. FI Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs (n=12 cells per group) with or without LPS treatment at day 40. J Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without LPS treatment at day 40. KN Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without LPS treatment at day 40 (n=12 cells per group). Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

Reduced glutathione (GSH) is an important antioxidant helps to prevent and reduce oxidative stress by neutralizing free radicals, widely used in the treatment of various types of oxidative stress-related diseases, including neurodegenerative diseases, cardiovascular diseases, and diabetes [25]. Hence, we treated PLEKHM2-KO hiPSC-CMs with GSH at day 30 to observe whether it could rescue the disease phenotype caused by PLEKHM2 deficiency. We found that PLEKHM2-KO hiPSC-CMs treated with GSH exhibited a considerable reduction in ROS levels (Fig. 7A, B) and significantly elevation of m level compared to untreated PLEKHM2-KO hiPSC-CMs (Fig. 7C, and Supplementary Fig. 7A). This indicates that inhibiting ROS helps improve mitochondrial function by preventing oxidative stress-induced damage to nearby mitochondria. Next, we observed the effects of GSH on the calcium transient and myocardial contractility of PLEKHM2-KO hiPSC-CMs. After GSH treatment, the diastolic calcium concentration and recovery velocity of PLEKHM2-KO hiPSC-CMs were comparable to that of WT hiPSC-CMs (Fig. 7D-H, and Supplementary Fig. 7B). Similarly, after GSH treatment, the PLEKHM2-KO hiPSC-CMs also showed significant improvements in contractile force (Fig. 7IM). These results further suggested the critical role of oxidative stress in mediating the disease phenotype of PLEKHM2-deficient cardiomyopathy.

A, B Representative flow cytometry analysis and quantification of cellular ROS levels showed that underwent GSH treatment PLEKHM2-KO hiPSC-CMs exhibited a considerable reduction in ROS levels comparable to WT hiPSC-CMs (n=4 independent experiments). C Quantification of JC-1 revealed that GSH treatment increased significantly the m level in PLEKHM2-KO hiPSC-CMs (more than 120 cells per group). D Representative line scan images of calcium transients of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40. EH Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40 (n=12 cells per group). I Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40. JM Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40 (n=12 cells per group). Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

Previous studies have shown that PLEKHM2 deficiency lead to abnormal lysosomal localization and impaired autophagic flux [2], causing damaged mitochondrial accumulation and ROS production. mTORC1 signaling pathway is the main negative regulator of autophagy, inhibiting autophagy by phosphorylating and inactivating key autophagy proteins such as ULK1 and ATG13 [26]. Hence, we investigated whether inhibition of mTORC1 by RAPA could boost autophagy and rescue the mitochondrial dysfunction and ROS generation in PLEKHM2-KO hiPSC-CMs. We found that p-mTOR levels were significantly higher in PLEKHM2-KO hiPSC-CMs than WT hiPSC-CMs (Supplementary Fig. 8A, B). Administration of RAPA significantly reduced the p-mTOR levels in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 8A, B). We further observed the effect of RAPA on autophagic flux in PLEKHM2-KO hiPSC-CMs. RAPA increased the number of autophagosomes (GFP+/RFP+ puncta) and autophagolysosome (GFP-/RFP+ puncta) in PLEKHM2-KO hiPSC-CMs, indicating that RAPA induced autophagy (Supplementary Fig. 8C). However, the number and proportion of GFP-/RFP+ puncta in PLEKHM2-KO hiPSC-CMs was still significantly lower than that WT hiPSC-CMs (Supplementary Fig. 8C), indicating that rapamycin cannot completely improve obstruction of autophagic flux caused by PLEKHM2 deficiency. We then evaluated the effects of RAPA treatment on mitochondrial function and myocardial contractility in PLEKHM2-KO hiPSC-CMs at day 40. We found that RAPA treatment partially improved m level and reduced ROS generation in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 8EG). Next, we observed the effects of RAPA treatment on myocardial contraction and calcium transient in PLEKHM2-KO hiPSC-CMs. We found that RAPA treatment enhanced the calcium transient amplitude (Supplementary Fig. 8HL) of PLEKHM2-KO hiPSC-CMs, but the myocardial contractility (Supplementary Fig. 8MR) was still significantly lower than those in WT hiPSC-CMs. These results indicated that administering RAPA cannot completely correct impaired autophagy caused by PLEKHM2 deficiency, but partially improves the disease phenotype of PLEKHM2-deficient cardiomyopathy.

We next investigated whether PLEKHM2-WT overexpression could restore autophagic flux in PLEKHM2-KO hiPSC-CMs and rescued the disease phenotype of PLEKHM2-deficient cardiomyopathy. We found that PLEKHM2-WT overexpression corrected the abnormal lysosomal localization (Supplementary Fig. 9) and increased the number and proportion of GFP-/RFP+ puncta in PLEKHM2-KO hiPSC-CMs, compared to untreated PLEKHM2-KO hiPSC-CMs (Fig. 8A, B). This indicates that PLEKHM2-WT overexpression improve the autophagic degradation in PLEKHM2-KO hiPSC-CMs. We further observed the effects of PLEKHM2-WT overexpression on the mitochondrial function of PLEKHM2-KO hiPSC-CMs. PLEKHM2-KO hiPSC-CMs treated with PLEKHM2-WT overexpression exhibited a significant increase in the m level and decrease in ROS levels compared to untreated PLEKHM2-KO hiPSC-CMs (Fig. 8CF). Subsequently, we evaluated the effects of PLEKHM2-WT overexpression on the calcium transient and myocardial contractility of PLEKHM2-KO hiPSC-CMs. PLEKHM2-WT overexpression significantly enhanced calcium transient amplitude (Fig. 8GK) and myocardial contractility (Fig. 8LP) compared to untreated PLEKHM2-KO hiPSC-CMs. This further demonstrates that PLEKHM2 plays a crucial role in regulating autophagy and clearing damaged mitochondria.

A, B Autophagic flux was assessed in hiPSC-CMs using mRFP-EGFP-LC3 adenovirus. Representative images and quantification of GFP and RFP+ puncta are shown in (A) and (B). C, D Representative immunofluorescence staining and and quantitative analysis of JC-1 revealed that PLEKHM2-WT overexpression ameliorated m of PLEKHM2-KO hiPSCs-CMs (more than 70 cells per group). Scale bars, 10 m. E, F Representative flow cytometry analysis and quantification of cell reactive oxygen species (ROS) intensity demonstrated PLEKHM2-WT overexpression reduced ROS levels of PLEKHM2-KO hiPSCs-CMs. G Representative line scan images of calcium transients of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40. HK Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40 (n=12 cells per group). L Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40. MP Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40 (n=12 cells per group). Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S. not significant.

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PLEKHM2 deficiency induces impaired mitochondrial clearance and elevated ROS levels in human iPSC-derived ... - Nature.com

Characterization of human iPSC-derived sensory neurons and their functional assessment using multi electrode array … – Nature.com

Characterization of the expression of hiPSC-derived sensory neurons

HiPSC-derived sensory neurons (cat #RCDN004N, Reprocell.Inc) were used in this study. To identify characteristics of human iPSC-derived sensory neurons, we confirmed sensory neuron-related genes and proteins expression by real-time PCR and immunocytochemistry (ICC). Real-time PCR shows an increase in Peripherin, Brn3a, TRPV1, TRPM8, Nav1.7, Nav1.8, Piezo2, TRKA, TRKC, TRKB, P2X3, H1R, MrgprX1, CGRP and TAC1 compared with hiPSC cultured 14days in vitro (DIV) (Fig.1). The nociceptor phenotype consists of A-fibers, and C-fibers. C-fibers respond to both peptidergic and non-peptidergic neurotransmitters. HiPSC-derived sensory neurons expressed TRKA (nociceptor marker), IB4 (A-fibers marker), CGRP and TAC1 (peptidergic neurotransmitters) and P2X3 (ATP (non-peptidergic neurotransmitter) receptor) (Figs. 1i,l,o,p and 2k). Furthermore, TRPV1, TRPM8, Nav1.7 and Nav1.8 which are nociceptors receptors were also expressed (Fig.1c,eg). TRPV1 is known to be activated by capsaicin and noxious heat (43C)14. TRPM8 is known to be activated by menthol, noxious (<15C) and non-noxious (2815C) heat15,16. Nav1.7 and Nav1.8 are known to be subtype of voltage-gated sodium channels which is preferentially expressed in nociceptors17,18,19. The mechanoreceptor phenotype consists of relatively large diameter cells that are A-fibers. HiPSC-derived sensory neurons expressed TRKB (mechanoreceptor marker), NF200 (A-fibers marker), TRPM8 and Piezo2 (mechanoreceptor receptors) (Figs. 1e,h,j and 2j)4. TRKC, a proprioceptor marker was expressed in hiPSC-derived sensory neurons (Fig.1k). Thus, these data suggests that the hiPSC-derived sensory neurons generated constitute a heterogeneous population of sensory neuronal subclasses. The expression of TRPA1 in hiPSC-derived sensory neurons was lower than the one in hiPSC (Fig.1d). The expression levels of Brn3a, TRPM8, TRKB and MrgprX1 in hiPSC-derived sensory neurons were comparable to those in human DRG, whereas the others were lower than in hDRG. The reason some genes of hiPSC-derived sensory neurons showed lower expression than hDRG might be due to the immature nature of the hiPSC-derived sensory neurons20. Although we cultured them for a long time, the expression levels of Peripherin, TRPV1, TRPA1, Nav1.7, Nav1.8, H1R, and CGRP were not comparable to the ones in hDRG (Supplementary Fig. S1). Therefore, we confirmed proteins expression by ICC.

Expression of sensory neuron related genes in hiPSC, hiPSC-derived sensory neurons and human DRG. Real-time PCR showed expression of (a) Peripherin, (b) Brn3a, (c) TRPV1, (d) TRPA1, (e) TRPM8, (f) Nav1.7, (g) Nav1.8, (h) Piezo2, (i) TRKA, (j) TRKB, (k) TRKC, (l) P2X3, (m) H1R, (n) MrgprX1, (o) CGRP, (p)TAC1. The square marker, the circle marker and triangle marker indicate expression of genes in hiPSC, hiPSC-derived sensory neurons and human DRG respectively. Three different lot of hiPSC-derived sensory neurons were examined. The line marker represents the mean expression of genes in hiPSC-derived sensory neurons.

Expression of sensory neuron related proteins in hiPSC-derived sensory neurons and their morphology. The cells are stained for (a) TUBB3, (b) Peripherin, (c) Brn3a, (d) TRPV1, (e) TRPM8, (f) Nav1.7, (g) TRKA, (h) TRKB, (i) TRKC, (j) NF200, (k) IB4. DAPI stain of nuclei is shown in blue. (l) Image of iPSC-derived sensory neurons which exhibit a bipolar (red arrowhead), pseudounipolar (yellow arrowhead), or multipolar morphology (green arrowhead). Scale bar represents 50m.

ICC showed expression of TUBB3 (mature neuron marker), Peripherin (peripheral neuron marker) and Brn3a (sensory neuron marker) at 14 DIV (Fig.2ac). TRPV1, TRPM8, Nav1.7, TRKA, TRKB and TRKC were expressed at the membrane (Fig.2di). Since TRPV1, and TRPM8 are receptors of noxious and non-noxious stimulation and are expressed at the membrane, we expected them to be available for characterizing their function using MEA (Fig.2d,e). NF200, a A-fibers marker, was expressed at a higher-intensity in relatively large diameter cells than small diameter cells (Fig.2j). Although adult human DRG do not bind IB4 which is a non-peptidergic C-fibers marker, it was expressed in hiPSC-derived sensory neurons (Fig.2k)21. The research showed expression of IB4 in prenatal human DRG at 8-month of gestation22. This data suggests that hiPSC-derived sensory neurons might be immature.

It is known that when observing rat DRG cells in the early stages of development, their morphology changes from bipolar cells to pseudounipolar cells23. Our hiPSC-derived sensory neurons exhibit a bipolar, pseudounipolar and multipolar morphology (Fig.2l). A majority of the hiPSC-derived sensory neurons were bipolar neurons. This image suggests that our hiPSC-derived sensory neurons contained neurons with different degrees of maturity.

Taken together, hiPSC-derived sensory neurons express sensory neuron-related genes and proteins. They constitute a heterogeneous population of nociceptors, mechanoreceptors, and proprioceptors, and they differ in maturity. Thus, we proceeded to characterize their function next.

We confirmed whether hiPSC-derived sensory neurons responded to capsaicin, menthol, noxious heat (4346C), which are noxious stimuli, and bradykinin, and non-noxious heat (3742C), which is a non-noxious stimulus14,15,16,24. Sensory neurons of DRG are used as in vitro model of nociceptive response. DRG responded to 10nM, 100nM, 1M capsaicin25,26. We measured and compared data before and after drug treatment (Fig.3a). We measured the response to treatment with 100nM capsaicin which resulted in increase in Mean Firing Rate (MFR) and Number of Bursts (NOB), whereas vehicle treatment had no effect on them (Fig.3d,e). Capsaicin-evoked activity is known to be rapid in DRG25. This result showed that neural activity was evoked within 10s after treatment with capsaicin in hiPSC-derived sensory neurons as well (Fig.3b). To determine whether capsaicin-evoked neuronal activity is characteristic of hiPSC-derived sensory neuron, we treated with 100nM capsaicin in hiPSC-derived cortical neurons. As a result, hiPSC-derived cortical neurons did not respond to capsaicin (Fig.3ce). Moreover, we added 100nM AMG9810 which is a TRPV1 antagonist for 60min before treating with capsaicin. A response to capsaicin was not observed in the presence of AMG9810 (Fig. S2a,b). These data suggest that capsaicin-evoked activity occurred via TRPV1 in iPSC-derived sensory neurons. Thus, we can conclude that hiPSC-derived sensory neurons specifically respond to noxious stimulus and could be used in functional assays using MEA.

Capsaicin and menthol responsiveness using MEA. (a) Timeline of drug treatment. Baseline and dose response were recorded for 60s when treating with capsaicin or menthol. Capsaicin experiment raster plots of (b) hiPSC-derived sensory neurons and (c) hiPSC-derived cortical neurons. The triangle marker indicates the time of capsaicin addition. (d, h) Mean Firing Rate normalized to the control. Control firing rate is calculated as firing rate before adding vehicle or drug. (e, i) Number of Bursts normalized to the control. Menthol experiment raster plot of (f) hiPSC-derived sensory neurons and (g) hiPSC-derived cortical neurons. n=3 wells.

Menthol activates TRPM8 which is a nociceptive receptor. Since mouse and rat DRG respond to 10M and 100M menthol, we decided to treat with the same concentrations26,27. The high concentration of menthol resulted in suppressing spontaneous neural activity (Supplementary Fig. S3). This may be due in part to the higher expression of TRPM8 in hiPSC-derived sensory neurons than in human DRG (Fig.1e, Supplementary Fig. S1e). Treatment with 100nM menthol resulted in an increase in MFR and NOB in hiPSC-derived sensory neurons whereas hiPSC-derived cortical neurons did not respond to menthol (Fig.3fi). The data show that menthol got a response from nociceptive-like and non-nociceptive-like DRG neurons28. Since our hiPSC-derived sensory neurons responded to menthol, they may also include functionally non-nociceptive like neurons.

Bradykinin activates nociceptors and causes pain,24. Treatment with 100nM bradykinin resulted in significant increase in MFR and NOB compared to vehicle treatment for 60s (n=3, p<0.05) (Fig.4ac). In contrast to capsaicin and menthol, the onset of bradykinin-evoked neural activity was relatively long (Figs. 3b,f and 4a). Bradykinin-evoked activity increased gradually and reached its mean peak at 60s in DRG25. However, hiPSC-derived sensory neurons were able to respond faster than DRG, because they also responded to an additional stimulation which immediately activated them when bradykinin and vehicle were added against the well of the MEA plate. There is expression of TRKB and Piezo2 relevant to touch sensation in hiPSC-derived sensory neurons, explaining why they may have responded to an additional stimulation.

Bradykinin responsiveness. (a) Bradykinin experiment raster plot of hiPSC-derived sensory neurons. The triangle marker indicates the time of bradykinin addition. (b) Mean Firing Rate after addition of Bradykinin normalized to firing rate before addition of Bradykinin. (c) Number of Bursts after addition of Bradykinin normalized to number of bursts before addition of Bradykinin. n=3 wells, *p<0.05.

MFR were observed to increase gradually in DRG, when temperature increases from 37 to 42C via the stage plate heater, part of the recording system25. We increased the temperature from 37 to 46C via MAESTROs system to confirm responsiveness to noxious heat and non-noxious heat. We observed that MFR and NOB increased gradually and reached their mean peak at 45C and 46C respectively, in hiPSC-derived sensory neurons (Fig.5). In the presence of TRPV1 antagonist, AMG9810, MFR were lower than that of vehicle at 4346C (Fig. S2c,d). Because TRPV1 is known to be activated by noxious heat (43C), these results suggest that TRPV1 may contribute to the response to 4346C in iPSC-derived sensory neurons. The relative levels of MFR (1.540.046) and NOB (1.620.01) at 41C, which is non-noxious heat, were significantly higher than those at 37C. However, MFR decreased gradually in hiPSC-derived cortical neurons with an increase in temperature (Fig.5b,c).

Temperature responsiveness. Raster plots of (a) hiPSC-derived sensory neurons and (b) hiPSC-derived cortical neurons when the temperature is gradually increased from 37 to 46C. (c) Mean Firing Rate normalized to the firing rate at 37C. (d) Number of Bursts normalized to the number of bursts at 37C. The data for the number of bursts in hiPSC-derived cortical neurons isnt shown because one of the three wells didnt produce any burst. n=3 wells, *p<0.05, **p<0.01 compared with the corresponding value at 37C. Functional assessment of hiPSC-derived sensory neurons against itching stimuli

These data suggest that the observed and recorded response is specific to sensory neurons and the hiPSC-derived sensory neuron populations generated in this study are likely to include nociceptors that respond to noxious stimuli like capsaicin, menthol, bradykinin, and noxious heat (43C) and to include mechanoreceptors that respond to non-noxious stimuli (41C).

Atopic dermatitis (AD) is the most common chronic skin disease which causes a global disease burden29. AD causes itch (pruritus) and poor non-health-based quality-of-life. It is known that itch occurs via C-fiber in nociceptors30. Recently, investigating itch has been established by using human sensory neurons from stem and other progenitor cells as in vitro model31. Although substances causing itch treat to their cells, their inhibitors effect arent confirmed using hiPSC-derived sensory neurons. Because we demonstrated that our hiPSC-derived sensory neurons expressed nociceptor genes and proteins, and responded to noxious stimuli, we expected that they also responded to an itch stimulus and its inhibitor.

Histamine is one of the substances that cause itching via C-fiber. The histamine receptor is a four G protein-coupled receptor. Histamine H1 receptor (H1R) is involved in the induction of histamine-induced pruritus32. Since we confirmed that the H1R gene is expressed, we examined whether hiPSC-derived sensory neurons respond to histamine, using MEA. Mouse DRG responded to 100M Histamine, as described in the literature33. HiPSC-derived sensory neurons didnt respond to 100M Histamine but responded to 1mM Histamine (Supplementary Fig. S3df and Fig.6a,b). The MFR gradually increased and reached its mean peak at 25min. Pyrilamine is a histamine H1 receptor inverse agonist. We treated the sensory neuron population with 10M Pyrilamine for 60min before adding Histamine. As a result, Pyrilamine inhibited Histamine-evoked activity (Fig.6a,b). These results suggest that Histamine-evoked activity occurred via H1R in iPSC-derived sensory neurons.

Histamine, H1R inhibitor, pyrilamine and chloroquine responsiveness. (a) The left raster plots have been recorded before histamine addition. The right raster plots have been recorded 25min after histamine addition. Upper raster plots are recorded in pyrilamine absence. Lower raster plots are recorded with presence of pyrilamine. (b, e) Mean Firing Rate normalized to firing rate before drug addition. Raster plots of (c) before chloroquine addition and (d) 5min after chloroquine addition. The experiment with histamine and pyrilamine was performed with n=2 wells each. Experiment with chloroquine was performed with n=3 wells. *p<0.05, **p<0.01 compared with the value recorded before addition or with vehicle.

Chloroquine is a drug that has been used in the treatment to prevent malaria. Histamine-independent pruritus is known to be one of the side effects of chloroquine34. Mrgprs are receptors of chloroquine and are activated by it35. Since we confirmed expression of human MrgprX1 by real-time PCR, we investigated the potential response of hiPSC-derived sensory neurons by chloroquine. DRG are reported to respond to 1mM chloroquine, however the MFR gradually decreased at the same concentration in hiPSC-derived sensory neurons (Supplementary Fig. S3g,h)36. 1M chloroquine increased the MFR and reached the mean peak after 5min of incubation (Fig.6ce). The mean peak after stimulation with chloroquine was reached faster than after stimulation with histamine.

These data showed an example of the effect of an itch inhibitor and different responses between itch inducing drugs. HiPSC-derived sensory neurons may be available for drug discovery against AD.

Nav1.7 subtype of voltage-gated sodium channels is expressed in DRG. Mutations in the gene encoding Nav1.7 are associated with either absence of pain or with exacerbation of pain. Recently, Nav1.7 has been an attractive target to pursue treating pain. ProTx-II is a tarantula venom peptide that preferentially inhibits Nav1.7 over other Nav subtypes37. It suppressed spontaneous neural activity in a time dependent manner in hiPSC-derived sensory neurons (Fig.7ae). The MFR and NOB are significantly diminished after 35min of incubation with 1M ProTx-II (Fig.7d,e). After washing out ProTx-II, the suppressed neural activity gradually recovered. Although, responses was completely blocked by 300nM ProTx-II in rodent DRG, the responses in hiPSC-derived sensory neurons were not blocked at the same concentration38. This may be due in part to the lower expression of Nav1.7 in hiPSC-derived sensory neurons than in human DRG.

Nav1.7 channels inhibitor, ProTx-II and Nav1.7 inhibitor responsiveness. Raster plots of (a) before ProTx-II addition (baseline), (b) 35min after adding ProTx-II and (c) 150min after washing ProTx-II, respectively. (d, i) Mean Firing Rate and (e, j) Number of Bursts normalized to mean firing rate and number of bursts before drug addition. Raster plots of (f) before Nav1.7 inhibitor addition (baseline), (b) 50min after adding Nav1.7 inhibitor and (c) 30min after washing Nav1.7 inhibitor, respectively. n=3 wells, *p<0.05, **p<0.01, ***p<0.001 compared to the value recorded before drug addition.

ProTx-II is known to act not only as a Nav1.7 inhibitor but also to act on Nav1.5 channels and on some T-Type Ca2+ channels39,40. Thus we administered a small molecule inhibitor that is more selective for Nav1.741. Although 300nM of a more selective Nav1.7 inhibitor suppressed MFR and NOB in a time dependent manner in hiPSC-derived sensory neurons, the degree of decrease was lower than that of ProTx-II (Fig.7fj). After washing it out, the suppression of the neural activity was lifted.

These results suggested that hiPSC-derived sensory neurons may serve as a drug screening tool for pain.

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Imaging cAMP nanodomains in human iPSC-derived cardiomyocytes – Nature.com

Cardiac activity is regulated by the -adrenergic pathway. The activation of this pathway triggers a cellular signalling cascade that increases the production of cAMP, a cyclic nucleotide that activates the enzyme protein kinase A (PKA). PKA phosphorylates key proteins involved in cellular contraction, but can also phosphorylate a multitude of other proteins with different functions. To achieve specific effects, cAMP is confined in nanoscale subcellular domains (nanodomains) close to PKA and its targets. The maintenance and regulation of these nanodomains are central to functional signal transduction, and their dysregulation can result in diseases such as heart failure.

I use this technique in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to study how the maturation of these cells is affected by a newly identified cAMP nanodomain found at gap junctions, which regulate the communication between adjacent cardiomyocytes. To understand the role of the gap junction-associated cAMP nanodomain in human iPSC-derived cardiomyocytes, endogenous levels of protein expression must be maintained to avoid interference with their maturation process. This technique can more broadly be used to study cAMP nanodomains in which overexpression of the target protein might impair cell physiology. This tool will provide unique insights into the processes involved in human iPSC-derived cardiomyocyte maturation and can also be used to identify new targets in the -adrenergic pathway that might be relevant for the treatment of diseases, such as heart failure.

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Imaging cAMP nanodomains in human iPSC-derived cardiomyocytes - Nature.com

Colossal Creates Elephant Stem Cells for the First Time in Quest to Revive the Woolly Mammoth – Singularity Hub

The last woolly mammoth roamed the vast arctic tundra 4,000 years ago. Their genes still live on in a majestic animal todaythe Asian elephant.

With 99.6 percent similarity in their genetic makeup, Asian elephants are the perfect starting point for a bold plan to bring the mammothor something close to itback from extinction. The project, launched by biotechnology company Colossal in 2021, raised eyebrows for its moonshot goal.

The overall playbook sounds straightforward.

The first step is to sequence and compare the genomes of mammoth and elephant. Next, scientists will identify the genes behind the physical traitslong hair, fatty depositsthat allowed mammoths to thrive in freezing temperatures and then insert them into elephant cells using gene editing. Finally, the team will transfer the nucleuswhich houses DNAfrom the edited cells into an elephant egg and implant the embryo into a surrogate.

The problem? Asian elephants are endangered, and their cellsespecially eggsare hard to come by.

Last week, the company reported a major workaround. For the first time, they transformed elephant skin cells into stem cells, each with the potential to become any cell or tissue in the body.

The advance makes it easier to validate gene editing results in the lab before committing to a potential pregnancywhich lasts up to 22 months for elephants. Scientists could, for example, coax the engineered elephant stem cells to become hair cells and test for gene edits that give the mammoth its iconic thick, warm coat.

These induced pluripotent stem cells, or iPSCs, have been especially hard to make from elephant cells. The animals are a very special species and we have only just begun to scratch the surface of their fundamental biology, said Dr. Eriona Hysolli, who heads up biosciences at Colossal, in a press release.

Because the approach only needs a skin sample from an Asian elephant, it goes a long way to protecting the endangered species. The technology could also support conservation for living elephants by providing breeding programs with artificial eggs made from skin cells.

Elephants might get the hardest to reprogram prize, said Dr. George Church, a Harvard geneticist and Colossal cofounder, but learning how to do it anyway will help many other studies, especially on endangered species.

Nearly two decades ago, Japanese biologist Dr. Shinya Yamanaka revolutionized biology by restoring mature cells to a stem cell-like state.

First demonstrated in mice, the Nobel Prize-winning technique requires only four proteins, together called the Yamanaka factors. The reprogrammed cells, often derived from skin cells, can develop into a range of tissues with further chemical guidance.

Induced pluripotent stem cells (iPSCs), as theyre called, have transformed biology. Theyre critical to the process of building brain organoidsminiature balls of neurons that spark with activityand can be coaxed into egg cells or models of early human embryos.

The technology is well-established for mice and humans. Not so for elephants. In the past, a multitude of attempts to generate elephant iPSCs have not been fruitful, said Hysolli.

Most elephant cells died when treated with the standard recipe. Others turned into zombie senescent cellsliving but unable to perform their usual biological functionsor had little change from their original identity.

Further sleuthing found the culprit: A protein called TP53. Known for its ability to fight off cancer, the protein is often dubbed the genetic gatekeeper. When the gene for TP53 is turned on, the protein urges pre-cancerous cells to self-destruct without harming their neighbors.

Unfortunately, TP53 also hinders iPSC reprogramming. Some of the Yamanaka factors mimic the first stages of cancer growth which could cause edited cells to self-destruct. Elephants have a hefty 29 copies of the protector gene. Together, they could easily squash cells with mutated DNA, including those that have had their genes edited.

We knew p53 was going to be a big deal, Church told the New York Times.

To get around the gatekeeper, the team devised a chemical cocktail to inhibit TP53 production. With a subsequent dose of the reprogramming factors, they were able to make the first elephant iPSCs out of skin cells.

A series of tests showed the transformed cells looked and behaved as expected. They had genes and protein markers often seen in stem cells. When allowed to further develop into a cluster of cells, they formed a three-layered structure critical for early embryo development.

Weve been really waiting for these things desperately, Church told Nature. The team published their results, which have not yet been peer-reviewed, on the preprint server bioRxiv.

The companys current playbook for bringing back the mammoth relies on cloning technologies, not iPSCs.

But the cells are valuable as proxies for elephant egg cells or even embryos, allowing the scientists to continue their work without harming endangered animals.

They may, for example, transform the new stem cells into egg or sperm cellsa feat so far only achieved in micefor further genetic editing. Another idea is to directly transform them into embryo-like structures equipped with mammoth genes.

The company is also looking into developing artificial wombs to help nurture any edited embryos and potentially bring them to term. In 2017, an artificial womb gave birth to a healthy lamb, and artificial wombs are now moving towards human trials. These systems would lessen the need for elephant surrogates and avoid putting their natural reproductive cycles at risk.

As the study is a preprint, its results havent yet been vetted by other experts in the field. Many questions remain. For example, do the reprogrammed cells maintain their stem cell status? Can they be transformed into multiple tissue types on demand?

Reviving the mammoth is Colossals ultimate goal. But Dr. Vincent Lynch at the University of Buffalo, who has long tried to make iPSCs from elephants, thinks the results could have a broader reach.

Elephants are remarkably resistant to cancer. No one knows why. Because the studys iPSCs are stripped of TP53, a cancer-protective gene, they could help scientists identify the genetic code that allows elephants to fight tumors and potentially inspire new treatments for us as well.

Next, the team hopes to recreate mammoth traitssuch as long hair and fatty depositsin cell and animal models made from gene-edited elephant cells. If all goes well, theyll employ a technique like the one used to clone Dolly the sheep to birth the first calves.

Whether these animals can be called mammoths is still up for debate. Their genome wont exactly match the extinct species. Further, animal biology and behavior strongly depend on interactions with the environment. Our climate has changed dramatically since mammoths went extinct 4,000 years ago. The Arctic tundratheir old homeis rapidly melting. Can the resurrected animals adjust to an environment they werent adapted to roam?

Animals also learn from each other. Without a living mammoth to show a calf how to be a mammoth in its natural habitat, it may adopt a completely different set of behaviors.

Colossal has a general plan to tackle these difficult questions. In the meantime, the work will help the project make headway without putting elephants at risk, according to Church.

This is a momentous step, said Ben Lamm, cofounder and CEO of Colossal. Each step brings us closer to our long-term goals of bringing back this iconic species.

Image Credit: Colossal Biosciences

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Colossal Creates Elephant Stem Cells for the First Time in Quest to Revive the Woolly Mammoth - Singularity Hub

How stem cells might be used in planned de-extinction of woolly mammoth – Cosmos

Sometimes it takes the smallest thing to undertake a mammoth task.

Thats what researchers behind the attempts to de-extinct the woolly mammoth are hoping as they announced what they believe to be a step forward in their efforts.

One Texas-based company has made de-extinction its business. It is looking at not only de-extinction of the woolly mammoth, but also dodos and Australias thylacine both hunted to extinction within the last 500 years.

The key is an announcement this week from researchers with Colossal Biosciences who say theyve derived induced pluripotent stem cells (iPSCs) from Asian elephants (Elephas maximus).

These iPSCs are reprogrammed to be able to give rise to any cell type in the body.

It means the researchers might be able to investigate the genetic differences between the woolly mammoth (Mammathus primigenius) and their closest living relatives the Asian elephant. They can also test gene edits without needing tissue from living animals.

Woolly mammoths roamed Earth for nearly 800,000 years.

They diversified from the steppe mammoth (Mammuthus trogontherii) at the beginning of the Middle Pleistocene (770,000126,000 years ago). They were closely related to the North American Columbian mammoth (Mammathus columbi) and DNA studies show they occasionally interbred.

Woolly mammoths were up to 3.5 metres tall at the shoulder and could weigh as much as 8 metric tons. (By contrast the Asian elephant is 2-3m and weights up to 5t.)

Mammoths are synonymous with the last Ice Age which ended about 12,000 years ago. Its believed that a combination of a warming climate and human hunting saw woolly mammoth numbers decline.

They died out so recently that some mammoth bodies have been recovered extremely well preserved in ice and snow.

The last stronghold of the woolly mammoth was the Siberian island of Wrangel where they lived until as recently as 4,000 years ago.

When these last mammoths died, the Great Pyramids of Giza were already 600 years old. Stonehenge had been around for 1,000 years and Sumerian poets had begun compiling the works that would over the next 800 years be brought together into the Epic of Gilgamesh.

In the great scheme of geological time, we are tantalisingly close to these remarkable creatures.

The successful formation of Asian elephant iPSCs in the lab is critical to understanding how the woolly mammoths genetic code sets it apart from its modern counterparts.

Which bits of DNA come together to produce features like their shaggy hair, curved tusks, fat deposits and dome skulls? These are the kinds of questions scientists at Colossal now feel they can answer.

It is also possible that the iPSCs can lead to producing elephant sperm and egg cells in the lab. Anyone whos had the birds and the bees chat doesnt need to be told why thats important in de-extinction.

Being able to create these cells in a lab is particularly important given the precariousness of Asian elephant populations.

Fewer than 50,000 Asian elephants remain in the wild according to the World Wildlife Fund. They are listed as endangered on the IUCNs Red List. Attempts to retrieve egg and sperm cells from Asian elephants would be difficult and potentially adverse.

Making elephant iPSCs has been so difficult because of complex gene pathways unique to these animals. Colossals genetic engineers overcame this by suppressing core genes called TP53 which regulate cell growth and halt the duplicating process.

But the work doesnt stop.

Theyre still looking at alternative methods to create iPSCs and maturing the ones theyve already made.

Theres also a lot still to learn about the complex 22-month gestation period of elephants if a healthy woolly mammoth calf is to be produced through in vitro fertilisation of a modern elephant.

Colossals plan is to have a living, breathing woolly mammoth by 2028.

For that to happen, the company is also looking into restoring suitable tundra steppe habitats in Canada and the US where the reborn mammoth population can settle.

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How stem cells might be used in planned de-extinction of woolly mammoth - Cosmos

The long and winding road of reprogramming-induced rejuvenation – Nature.com

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Continued here:
The long and winding road of reprogramming-induced rejuvenation - Nature.com

Looking for the Path to Safe Cell Rejuvenation – Lifespan.io News

In Nature Communications, Ali Ycel and Vadim Gladyshev have published a review of the current state of the art in partial cellular reprogramming, detailing what this technology does and how it might be used safely.

This paper begins by treading familiar ground on the subject, explaining its end goals and purpose. When successful, partial cellular reprogramming induces reprogramming-induced rejuvenation (RIR), a state in which a cell is transformed into an epigenetically younger cell of the same type and fulfilling the same function [1]. This process has had multiple crucial successes in experimental models, including human muscle cells [2] and skin cells [3] along with restoring vision [4] and extending lifespan [5] in mice.

Much of this work has been done in mice that have been genetically modified to express the necessary factors when doxycycline is administered. This has even been accomplished after birth via an adeno-associated virus (AAV) [5]. While there are four Yamanaka factors, OSKM, the fourth, c-Myc, is often omitted because it raises the risk of cancer. OSK administration significantly reduced the frailty of the treated mice.

As the authors note, applying these sorts of genetic modification techniques directly to human beings is currently infeasible with existing technologies. Partial reprogramming requires carefully determined generation of Yamanaka factors inside cells. To apply this in a clinical setting would require gene therapy that has specific and strong effects on individual tissues, and using the AAV system that works on mice is not yet practical for people [6]. Generating partially reprogrammed cells outside the body, similarly to how induced pluripotent stem cells (iPSCs) are generated, may be feasible for therapeutic purposes.

Administering small molecules to people in order to effect rejuvenation in the form of a drug has been the dream of aging researchers for some time. Previous work has spurred the creation of iPSCs through such chemical means [7]. The authors of this review describe these methods as less powerful than gene therapy and requiring multiple stages of administration. This implies a degree of safety and control that makes them more attractive for human research.

An experiment on mouse cells, which also included Vadim Gladyshev, had revealed that using a 7c cocktail reduced multiple aspects of aging, including epigenetic clock measurements, age-related metabolic changes, and oxidative stress markers [8]. However, it also upregulates the senescence-associated p53 pathway, which is downregulated through normal reprogramming methods and may cause cells to become senescent earlier [9].

Normally, constant expression of the Yamanaka factors in a living organism causes its cells to completely revert to a pluripotent state, in which they forget their roles, become cancerous, and cause the organism to die. For example, inducing OSKM for six days in the hearts of mice was found to be beneficial for them, while extending it for a dozen days proved lethal [10]. However, constantly inducing OSK in neural ganglion cells for a full 10-18 months improved vision without this side effect [4].

The authors note many of the aspects of aging that are improved or possibly improved with RIR, of which the most obvious, epigenetic alterations, is only one. Inflammation and proteostasis are also affected. Telomere attrition, however, occurs only in later reprogramming and is not affected by the partial variety [11]. Direct changes to cellular communication and genomic stability are not yet known.

However, the authors point out that, while full reprogramming does not cells to mutate, creating colonies of iPSCs causes evolutionary pressure: cells with mutations that may not be beneficial for the whole organism may be more prevalent in iPSC colonies [12]. It remains to be seen if this is a concern for partial reprogramming.

The authors also mention a biochemical pluripotency network and the fundamental differences between full and partial rejuvenation. Most critically, they hold that partial reprogramming is caused by factors that are downstream of full reprogramming. If it is possible to directly affect these factors instead of relying on the Yamanaka full-reprogramming factors, it might be possible to cause RIR without risking the dangerous side effects associated with complete reprogramming. However, this area of research remains unexplored.

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[1] Ocampo, A., Reddy, P., Martinez-Redondo, P., Platero-Luengo, A., Hatanaka, F., Hishida, T., & Belmonte, J. C. I. (2016). In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell, 167(7), 1719-1733.

[2] Sarkar, T. J., Quarta, M., Mukherjee, S., Colville, A., Paine, P., Doan, L., & Sebastiano, V. (2020). Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nature communications, 11(1), 1545.

[3] Gill, D., Parry, A., Santos, F., Okkenhaug, H., Todd, C. D., Hernando-Herraez, I., & Reik, W. (2022). Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. Elife, 11, e71624.

[4] Lu, Y., Brommer, B., Tian, X., Krishnan, A., Meer, M., Wang, C., & Sinclair, D. A. (2020). Reprogramming to recover youthful epigenetic information and restore vision. Nature, 588(7836), 124-129.

[5] Macip, C. C., Hasan, R., Hoznek, V., Kim, J., Lu, Y. R., Metzger IV, L. E., & Davidsohn, N. (2024). Gene Therapy-Mediated Partial Reprogramming Extends Lifespan and Reverses Age-Related Changes in Aged Mice. Cellular Reprogramming, 26(1), 24-32.

[6] Pupo, A., Fernndez, A., Low, S. H., Franois, A., Surez-Amarn, L., & Samulski, R. J. (2022). AAV vectors: The Rubiks cube of human gene therapy. Molecular Therapy.

[7] Guan, J., Wang, G., Wang, J., Zhang, Z., Fu, Y., Cheng, L., & Deng, H. (2022). Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature, 605(7909), 325-331.

[8] Mitchell, W., Goeminne, L. J., Tyshkovskiy, A., Zhang, S., Chen, J. Y., Paulo, J. A., & Gladyshev, V. N. (2023). Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation. bioRxiv, 2023-06.

[9] Tyner, S. D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H., & Donehower, L. A. (2002). p53 mutant mice that display early ageing-associated phenotypes. Nature, 415(6867), 45-53.

[10] Chen, Y., Lttmann, F. F., Schoger, E., Schler, H. R., Zelarayn, L. C., Kim, K. P., & Braun, T. (2021). Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science, 373(6562), 1537-1540.

[11] Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. cell, 126(4), 663-676.

[12] Kosanke, M., Osetek, K., Haase, A., Wiehlmann, L., Davenport, C., Schwarzer, A., & Martin, U. (2021). Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes. Molecular Therapy, 29(8), 2535-2553.

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Looking for the Path to Safe Cell Rejuvenation - Lifespan.io News