iPSC Reprogramming Research Articles

Establishment of two human induced pluripotent stem cell lines from familial long QT syndrome type 1 patients carrying KCNQ1 mutation

Long QT syndrome type 1 (LQT1) is a rare heart disorder caused by a loss-of-function mutation in the KCNQ1 gene that causes loss of Kv7.1 channel function, which can lead to Palpitations, Syncope, and Sudden cardiac arrest. We derived induced pluripotent stem cells from PBMC of LQT1 patients carrying a pathogenic variant (c.734G>A; p.Gly245Glu). The non-integrative Sendai virus-mediated iPSC reprogramming method was used for iPSC line generation. These iPSC cell lines exhibit stem cell pluripotency, differentiation capability, and cell morphology, resulting in a reliable cell source to study the effects of KCNQ1 mutation in disease-specific cell types.

Establishment of a human-induced pluripotent stem cell line from a long QT syndrome type 2 patient harboring a KCNH2 mutation

Long QT syndrome type 2 (LQT2) is a heart disorder resulting from a loss-of-function mutation in theKCNH2gene that causes loss of Kv11.1 channel function, potentially resulting in syncope, arrhythmias, and sudden death. We derived induced pluripotent stem cell line from PBMC of LQT2 patient carrying a variant of pathogenic variant (c.157G > A; p.Gly53Ser). The generation of iPSC lines was achieved using the non-integrative Sendai virus-mediated iPSC reprogramming method. The iPSC cell line exhibit pluripotency, normal karyotype, stem cell morphology, and differentiation capability, resulting a reliable cell source to study the effects of KCNH2 mutation in disease-specific cell types.

Early inhibition of BRD4 facilitates iPSC reprogramming via accelerating rDNA dynamic expression

BackgroundiPSC reprogramming technology exhibits significant promise in the realms of clinical therapeutics, disease modeling, pharmaceutical drug discovery, and various other applications. However, the extensive utilization of this technology has encountered impediments in the form of inefficiency, prolonged procedures, and ambiguous biological processes. Consequently, in order to improve this technology, it is of great significance to delve into the underlying mechanisms involved in iPSC reprogramming. The BET protein BRD4 plays a crucial role in the late stage of reprogramming; however, its precise function in the early stage remains unclear.ResultsOur study aims to investigate BRD4’s role in the early stages of iPSC reprogramming. Our investigation reveals that early inhibition of BRD4 substantially enhances iPSC reprogramming, whereas its implementation during the middle-late stage impedes the process. During the reprogramming, ribosome DNA expression initially increases before decreasing and then gradually recovers. Early inhibition of BRD4 improved the decline and restoration of rDNA expression in the early and middle-late stages, respectively. Additionally, we uncovered the mechanism of BRD4’s regulation of rDNA transcription throughout reprogramming. Specifically, BRD4 interacts with UBF and co-localizes to both the rDNA promoter and enhancer regions. Ultimately, BRD4 facilitates rDNA transcription by promoting the enrichment of histone H3 lysine 27 acetylation in the surrounding chromatin. Moreover, we also discovered that early inhibition of BRD4 facilitates cells’ transition out of the somatic cell state and activate pluripotent genes.ConclusionsIn conclusion, our results demonstrate that early inhibition of BRD4 promotes sequential dynamic expression of rDNA, which improves iPSC reprogramming efficiency.

RetSat stabilizes mitotic chromosome segregation in pluripotent stem cells

BackgroundChromosome stability is crucial for homeostasis of pluripotent stem cells (PSCs) and early-stage embryonic development. Chromosomal defects may raise carcinogenic risks in regenerative medicine when using PSCs as original materials. However, the detailed mechanism regarding PSCs chromosome stability maintenance is not fully understood.MethodsMouse embryonic stem cells (line D3) and human embryonic stem cells (line H9) were cultured under standard conditions. To confirm the loading of RetSat protein on mitotic chromosomes of PSCs, immunostaining was performed in PSCs spontaneous differentiation assay and iPSC reprogramming assay from mouse embryonic fibroblasts (MEFs), respectively. In addition, qPCR, immunoprecipitation, LC-MS/MS and immunoblotting were used to study the expression of RetSat, and interactions of RetSat with cohesin/condensin components. RNA sequencing and teratoma formation assay was conducted to evaluate the carcinogenic risk of mouse embryonic stem cells with RetSat deletion.ResultsWe reported a PSC high-expressing gene, RetSat, plays key roles in chromosome stabilization. We identified RetSat protein localizing onto mitotic chromosomes specifically in stemness positive cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found dramatic chromosome instability, e.g. chromosome bridging, lagging and interphase micronuclei in mouse and human ESCs when down regulating RetSat. RetSat knock-out mouse ESCs upregulated cancer associated gene pathways, and displayed higher tumorigenic capacities in teratoma formation assay. Mechanistically, we confirmed that RetSat interacts with cohesin/condensin components Smc1a and Nudcd2. RetSat deletion impaired the chromosome loading dosage of Smc1a, Smc3 and Nudcd2.ConclusionsIn summary, we reported RetSat to be a key stabilizer of chromosome condensation in pluripotent stem cells. This highlights the crucial roles of RetSat in early-stage embryonic development, and potential value of RetSat as an effective biomarker for assessing the quality of pluripotent stem cells.

The interferon γ pathway enhances pluripotency and X-chromosome reactivation in iPSC reprogramming.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here, we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

KIF1A, R1457Q, and P1688L Mutations Induce Protein Abnormal Aggregation and Autophagy Impairment in iPSC-Derived Motor Neurons.

Mutations in the C-terminal of KIF1A (Kinesin family member 1A) may lead to amyotrophic lateral sclerosis (ALS) through unknown mechanisms that are not yet understood. Using iPSC reprogramming technology and motor neuron differentiation techniques, we generated iPSCs from a healthy donor and two ALS patients with KIF1A mutations (R1457Q and P1688L) and differentiated them into spinal motor neurons (iPSC-MN) to investigate KIF1A-related ALS pathology. Our in vitro iPSC-iMN model faithfully recapitulated specific aspects of the disease, such as neurite fragmentation. Through this model, we observed that these mutations led to KIF1A aggregation at the proximal axon of motor neurons and abnormal accumulation of its transport cargo, LAMP1, resulting in autophagy dysfunction and cell death. RNAseq analysis also indicated that the functions of the extracellular matrix, structure, and cell adhesion were significantly disturbed. Notably, using rapamycin during motor neuron differentiation can effectively prevent motor neuron death.

Unlocking cellular plasticity: enhancing human iPSC reprogramming through bromodomain inhibition and extracellular matrix gene expression regulation.

The transformation from a fibroblast mesenchymal cell state to an epithelial-like state is critical for induced pluripotent stem cell (iPSC) reprogramming. In this report, we describe studies with PFI-3, a small-molecule inhibitor that specifically targets the bromodomains of SMARCA2/4 and PBRM1 subunits of SWI/SNF complex, as an enhancer of iPSC reprogramming efficiency. Our findings reveal that PFI-3 induces cellular plasticity in multiple human dermal fibroblasts, leading to a mesenchymal-epithelial transition during iPSC formation. This transition is characterized by the upregulation of E-cadherin expression, a key protein involved in epithelial cell adhesion. Additionally, we identified COL11A1 as a reprogramming barrier and demonstrated COL11A1 knockdown increased reprogramming efficiency. Notably, we found that PFI-3 significantly reduced the expression of numerous extracellular matrix (ECM) genes, particularly those involved in collagen assembly. Our research provides key insights into the early stages of iPSC reprogramming, highlighting the crucial role of ECM changes and cellular plasticity in this process.

Ultrasound-triggered three dimensional hyaluronic acid hydrogel promotes in vitro and in vivo reprogramming into induced pluripotent stem cells

Cellular reprogramming technologies have been developed with different physicochemical factors to improve the reprogramming efficiencies of induced pluripotent stem cells (iPSCs). Ultrasound is a clinically applied noncontact biophysical factor known for regulating various cellular behaviors but remains uninvestigated for cellular reprogramming. Here, we present a new reprogramming strategy using low-intensity ultrasound (LIUS) to improve cellular reprogramming of iPSCs in vitro and in vivo. Under 3D microenvironment conditions, increased LIUS stimulation shows enhanced cellular reprogramming of the iPSCs. The cellular reprogramming process facilitated by LIUS is accompanied by increased mesenchymal to epithelial transition and histone modification. LIUS stimulation transiently modulates the cytoskeletal rearrangement, along with increased membrane fluidity and mobility to increase HA/CD44 interactions. Furthermore, LIUS stimulation with HA hydrogel can be utilized in application of both human cells and in vivo environment, for enhanced reprogrammed cells into iPSCs. Thus, LIUS stimulation with a combinatorial 3D microenvironment system can improve cellular reprogramming in vitro and in vivo environments, which can be applied in various biomedical fields.

Knockdown H19 Accelerated iPSCs Reprogramming through Epigenetic Modifications and Mesenchymal-to-Epithelial Transition.

H19 is an essential imprinted gene that is expressed to govern normal embryonic development. During reprogramming, the parental pronuclei have asymmetric reprogramming capacities and the critical reprogramming factors predominantly reside in the male pronucleus. After inhibiting the expression of H19 and Gtl2, androgenetic haploid ESCs (AG-haESCs) can efficiently and stably support the generation of healthy SC pups at a rate of ~20%, and double-knockout parthenogenetic haESCs can also produce efficiently. Induced pluripotent stem (iPS) cell reprogramming is thought to have a characteristic epigenetic pattern that is the reverse of its developmental potential; however, it is unclear how H19 participates in iPS cell reprogramming. Here, we showed that the expression of H19 was transiently increased during iPSC reprogramming. H19 knockdown resulted in greater reprogramming efficiency. The genes associated with pluripotency showed enhanced expression during the early reprogramming process, and the Oct4 promoter was demethylated by bisulfite genomic sequencing analysis. Moreover, expression analysis revealed that the mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) were downregulated during reprogramming in H19 knockdown. These findings provide functional insight into the role of H19 as a barrier to the early reprogramming process.

Dux activates metabolism-lactylation-MET network during early iPSC reprogramming with Brg1 as the histone lactylation reader.

The process of induced pluripotent stem cells (iPSCs) reprogramming involves several crucial events, including the mesenchymal-epithelial transition (MET), activation of pluripotent genes, metabolic reprogramming, and epigenetic rewiring. Although these events intricately interact and influence each other, the specific element that regulates the reprogramming network remains unclear. Dux, a factor known to promote totipotency during the transition from embryonic stem cells (ESC) to 2C-like ESC (2CLC), has not been extensively studied in the context of iPSC reprogramming. In this study, we demonstrate that the modification of H3K18la induced by Dux overexpression controls the metabolism-H3K18la-MET network, enhancing the efficiency of iPSC reprogramming through a metabolic switch and the recruitment of p300 via its C-terminal domain. Furthermore, our proteomic analysis of H3K18la immunoprecipitation experiment uncovers the specific recruitment of Brg1 during reprogramming, with both H3K18la and Brg1 being enriched on the promoters of genes associated with pluripotency and epithelial junction. In summary, our study has demonstrated the significant role of Dux-induced H3K18la in the early reprogramming process, highlighting its function as a potent trigger. Additionally, our research has revealed, for the first time, the binding of Brg1 to H3K18la, indicating its role as a reader of histone lactylation.

Epigenomic states contribute to coordinated allelic transcriptional bursting in iPSC reprogramming.

Two alleles of a gene can be transcribed independently or coordinatedly, which can lead to temporal expression heterogeneity with potentially distinct impacts on cell fate. Here, we profiled genome-wide allelic transcriptional burst kinetics during the reprogramming of MEF to induced pluripotent stem cells. We show that the degree of coordination of allelic bursting differs among genes, and alleles of many reprogramming-related genes burst in a highly coordinated fashion. Notably, we show that the chromatin accessibility of the two alleles of highly coordinated genes is similar, unlike the semi-coordinated or independent genes, suggesting the degree of coordination of allelic bursting is linked to allelic chromatin accessibility. Consistently, we show that many transcription factors have differential binding affinity between alleles of semi-coordinated or independent genes. We show that highly coordinated genes are enriched with chromatin accessibility regulators such as H3K4me3, H3K4me1, H3K36me3, H3K27ac, histone variant H3.3, and BRD4. Finally, we demonstrate that enhancer elements are highly enriched in highly coordinated genes. Our study demonstrates that epigenomic states contribute to coordinated allelic bursting to fine-tune gene expression during induced pluripotent stem cell reprogramming.

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages.

Circadian regulation of gene expression is prevalent and plays critical roles in cell differentiation. However, its roles in the reprogramming of differentiated cells remain largely unknown. Here, we found that one of the master circadian regulators PER1 promoted virus-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to induced neurons (iNs) and induced pluripotent stem cells (iPSCs). Unexpectedly, PER1 achieved this by repressing inflammatory activation of contaminating macrophages in the MEF culture, rather than by directly modulating the reprogrammability of MEFs. More specifically, we found that transduced viruses activated inflammatory genes in macrophages, such as Tnf encoding TNFα, one of the central inflammatory regulators and an autocrine activator of macrophages. TNFα inhibited iN reprogramming, whereas a TNFα inhibitor promoted iN reprogramming, connecting the inflammatory responses to iN reprogramming. In addition, macrophages were induced to proliferate and mature by non-macrophage cells serving as feeders, which also supported up-regulation of TNFα in macrophages without virus transduction. Furthermore, the 2 inflammatory responses were repressed by the circadian regulator PER1 in macrophages, making reprogrammability dependent on time-of-day of virus transduction. Similar results were obtained with iPSC reprogramming, suggesting a wide occurrence of macrophage-mediated inhibition of cell reprogramming. This study uncovers mechanistic links between cell reprogramming, bystander inflammatory macrophages, and circadian rhythms, which are particularly relevant to in vivo reprogramming and organoid formation incorporating immune cells.

Abstract A041: Rejuvenation: Innovative technology to improve T-cell antitumor properties through partial reprogramming

Abstract The T-cell identity and age determine function and fitness over a T-cell’s lifespan. This is particularly relevant when T cells are derived from patients with chronic viral infection or cancer. It is well known that increased T-cell age and differentiation and increased effector and exhausted phenotypes are associated with reduced anti-tumor efficacy and the need for higher infusion T-cell numbers for the treatment of hematological or solid tumors during adoptive cell therapies (ACT) (Kishton 2022). In an effort to overcome these barriers, methods to de-differentiate T cells into induced pluripotent stem cells (iPSCs) that return to embryonic immaturity, but lose their functional identity, have been extensively explored in the past years. Early work revealed several challenges to re-differentiate iPSCs into T cells with the desired functional phenotype, requiring a complex and time-consuming process. Here, we bring our novel strategy that counters the impact of aging on T-cell function through cellular rejuvenation without de-differentiating to iPSCs. We achieved T-cell rejuvenation via partial reprogramming of aged T cells by transiently expressing transcription factors associated with iPSC reprogramming. This proprietary partial reprogramming methodology reduces epigenetic age and rejuvenates T cells while maintaining the phenotype and function of conventional T cells. We were the first to illustrate the ability to reduce the epigenetic age of T cells without fully de-differentiating to iPSCs. Our initial studies with PBMCs showed a significant reduction in epigenetic age. On subsequent RNAseq analyses, we observed that rejuvenated and conventional T cells have equivalent transcriptomes, suggesting the maintenance of identity. Functionally, the TRJ cells are characterized by greatly improved cell-expansion capacity, together with increased expression of markers associated with T-cell stemness, including CCR7 and CD62L. In vitro studies of NY-ESO-1-targeted T-cell receptor (TCR) or a CD19-targeted chimeric antigen receptor (CAR) TRJ cells exhibited improved antitumor properties compared with non-rejuvenated T-cell control (TCT) cells in sequential cell-killing assays. We also confirmed the enhanced in vivo antitumor efficiency of NY-ESO-1 TCR TRJ cells in a murine xenograft tumor model. When tumor-infiltrating lymphocytes (TILs) are rejuvenated with the same rejuvenation technology the TILs showed enhanced cell-expansion capacity, and improvements in T-cell stemness phenotype. These results suggest the potential application of T-cell rejuvenation across multiple adoptive T-cell therapeutic modalities and may potentially improve outcomes for patients with solid tumors. Citation Format: Jessica Fioravanti, Yasuhiro Yamazaki, Takuya Maeda, Yin Huang, Naritaka Tamaoki, Kriti Bahl, Burak Kutlu, Shobha Potluri, Gary Lee, Nicholas P Restifo, Raul Vizcardo. Rejuvenation: Innovative technology to improve T-cell antitumor properties through partial reprogramming [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A041.

Age‐equivalent induced neurons from patients reveal neuronal fate loss and metabolic transformation underlying neurodegeneration in sporadic Alzheimer’s disease

AbstractBackgroundOld age is the dominating risk factor for Alzheimer’s Disease (AD), which exclusively affects people at older ages. Sporadic AD represents the overwhelming majority of all cases, but most research on AD has been performed on genetic causes and their directly related pathways, also because we were in lack of models that can reflect complex human genetics and age in a neuronal context.MethodPatient‐specific iPSC models represent an attractive solution, but iPSC reprogramming results in cellular rejuvenation and thus yields phenotypically young neurons mirroring fetal development. By contrast, direct conversion of old patient fibroblasts into induced neurons (iNs) preserves endogenous signatures of aging and produces neurons that resemble the adult human brain on an epigenome‐ and transcriptome‐wide scale. To control for the involvement of aging in AD, we generated age‐equivalent fibroblast‐derived iNs and rejuvenated iPSC‐derived neurons from a cohort of AD patients and controls.ResultPatient‐derived AD iNs reflect a hypo‐mature neuronal identity characterized by markers of stress, cell cycle, glycolytic reprogramming, and de‐differentiation, which share similarities with malignant cancer transformation and age‐dependent epigenetic erosion. We identified an isoform switch of pyruvate kinase M (PKM) to the PKM2 isoform as a trigger of a Warburg effect‐like metabolic switch, and further as a key transcriptional regulator leading to cell fate instability. Metabolomics, glucose tracing, and acetylome analysis confirmed a hyper‐acetylated state of AD iNs. This affects several splicing factors, triggering an isoform switch to PKM that ultimately leads to a loss of neuronal fate and maturity. Inhibition of the resulting citrate shunt to acetylCoA modulates the isoform switch in favor of PKM1, and re‐instates a mature and more resilient neuronal state.ConclusionWe conclude that age‐equivalent adult‐like iNs from patients are a useful model to study age‐dependent pathological signatures of AD. This iN model identifies AD‐related neuronal changes as part of an active cellular program that impairs neuronal cell identity and neuronal resilience by utilizing cancer‐related signaling cascades. We further reason that the AD‐specific metabolic switch and fate loss in neurons regulates, and is reinforced by, alternative splicing of PKM.

Overcoming the Variability of iPSCs in the Manufacturing of Cell-Based Therapies

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor’s genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.

ICF1-Syndrome-Associated DNMT3B Mutations Prevent De Novo Methylation at a Subset of Imprinted Loci during iPSC Reprogramming.

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation.

Towards Early Prediction of Human iPSC Reprogramming Success

This paper presents advancements in automated early-stage prediction of the success of reprogramming human induced pluripotent stem cells (iPSCs) as a potential source for regenerative cell therapies. The minuscule success rate of iPSC-reprogramming of around 0.01% to 0.1% makes it labor-intensive, time-consuming, and exorbitantly expensive to generate a stable iPSC line since that requires culturing of millions of cells and intense biological scrutiny of multiple clones to identify a single optimal clone. The ability to reliably predict which cells are likely to establish as an optimal iPSC line at an early stage of pluripotency would therefore be ground-breaking in rendering this a practical and cost-effective approach to personalized medicine.<br>Temporal information about changes in cellular appearance over time is crucial for predicting its future growth outcomes. In order to generate this data, we first performed continuous time-lapse imaging of iPSCs in culture using an ultra-high resolution microscope. We then annotated the locations and identities of cells in late-stage images where reliable manual identification is possible. Next, we propagated these labels backwards in time using a semi-automated tracking system to obtain labels for early stages of growth. Finally, we used this data to train deep neural networks to perform automatic cell segmentation and classification.<br>Our code and data are available at <a href='https://github.com/abhineet123/ipsc_prediction'>https://github.com/abhineet123/ipsc_prediction</a>

Influence of sarcomeric protein mutations on myocardial function in a 3D-disease model of human iPSC-derived bioartificial cardiac tissue

Abstract Background Peripartum cardiomyopathy (PPCM) is a rare type of cardiomyopathy that occurs in women without prior history of heart disease at later stages of pregnancy or several months after childbirth (1). The disease is characterized by lowered left ventricular ejection fraction (LVEF<45%) and can lead to heart failure if left untreated (1). Approximately 20% of PPCM patients harbor sarcomeric mutations, from which titin truncating variants (TTNtv-s) have the highest pathogenic potential (2). However, the role of such genetic defects of the contractile apparatus on PPCM pathogenesis is not well understood. Purpose The study aims to establish an in vitro 3D disease model of PPCM to elucidate the role of sarcomeric mutations, including titin mutations, in its etiology. We hypothesize, that the disease phenotype can be uncovered in vitro via the stimulation of PPCM patient-derived Bioartificial Cardiac Tissues (BCTs), utilizing pregnancy-related factors in our custom-made bioreactors (3). Methods We selected three patients carrying previously unstudied mutations in the TTN gene for the study. The iPSC reprogramming was performed using a commercial, Sendai virus-based kit. Pluripotency was confirmed via flow cytometry and immunofluorescence stainings. The presence of mutations was confirmed by exome sequencing. Cardiomyocyte differentiation (4) and BCT generation (3) were carried out via established protocols. Following maturation, BCTs were stimulated with the pregnancy-related factors, and tissue forces were measured in the bioreactors. These forces include intrinsically generated (spontaneous) and electrically induced (paced) contraction forces (mN), as well as passive forces, which correlate with tissue stiffness. Results We successfully generated two clonal induced pluripotent stem cell (iPSC) lines from each patient. We confirmed that all of the cell lines were >90% positive for pluripotency markers. After differentiation, CMs contracted spontaneously from day 7 onwards and were >95% positive for cardiac markers (SA, MF20, cTnT). The BCTs remodeled and started contracting on days 1-2 after generation, similarly to wild-type tissues in the baseline conditions. Prolactin treatment of Patient A, harboring a missense mutation in titin, did not show significant functional effects. We confirmed, that patients B and C harbor TTNtvs, and tissues from these patients show a trend of altered stiffness after stimulation with repeated mechanical stretch cycles or β-adrenergic agonist isoprenaline. Further analysis of the underlying mechanisms investigating metabolic changes, signaling pathways, and transcriptomics are ongoing. Conclusion The established iPSC and tissue engineering technologies offer a valuable tool for the development of an iPS-CM-based 3D in vitro disease model for PPCM, which can be used for risk stratification of individual and combined sarcomeric mutations and the development of tailored therapeutic strategies.

Integrative Meta-Analysis during Induced Pluripotent Stem Cell Reprogramming Reveals Conserved Networks and Chromatin Accessibility Signatures in Human and Mouse

iPSC reprogramming involves dynamic changes in chromatin accessibility necessary for the conversion of somatic cells into induced pluripotent stem cells (iPSCs). IPSCs can be used to generate a wide range of cells to potentially replace damaged cells in a patient without the threat of immune rejection; however, efficiently reprogramming cells for medical applications remains a challenge, particularly in human cells. Here, we conducted a cross-species meta-analysis to identify conserved and species-specific differences in regulatory patterns during reprogramming. Chromatin accessibility and transcriptional data as fibroblasts transitioned to iPSCs were obtained from the publicly available Gene Expression Omnibus (GEO) database and integrated to generate time-resolved regulatory networks during cellular reprogramming. We observed consistent and conserved trends between the species in the chromatin accessibility signatures as cells transitioned from fibroblasts into iPSCs, indicating distal control of genes associated with pluripotency by master reprogramming regulators. Multi-omic integration showed key network changes across reprogramming states, revealing regulatory relationships between chromatin regulators, enhancers, transcription factors, and target genes that result in the silencing of the somatic transcription program and activation of the pluripotency gene regulatory network. This integrative analysis revealed distinct network changes between timepoints and leveraged multi-omics to gain novel insights into the regulatory mechanisms underlying reprogramming.