iPSC Colonies Research Articles

Effects of Clump Size on the Pluripotency and Proliferation in the Passaging Process of Mouse Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are a promising cell source because of their pluripotency and self-renewal abilities. However, there is a risk of pluripotency loss during cell expansion. Particularly, cell passaging is associated with a higher risk of decreasing cell quality. There are two iPSC passaging methods: single-cell and clump passaging. Single-cell passaging is a rapid and simple method for cell manipulation, whereas clump passaging is superior for maintaining iPSC pluripotency. Therefore, clump passaging is a robust method for expanding iPSCs while maintaining their pluripotency. However, clump size control during clump passaging is difficult because colony fragmentation is performed manually by pipetting the colonies detached from the cell culture substrates. In this study, the effect of pipetting on iPSC colony fragmentation was evaluated and the relationship between iPSC clump size and pluripotency was clarified. An automated pipetting device was developed to standardize the clump passage process. The effect of clump size on the pluripotency and proliferative capacity of mouse iPSCs was investigated. Clump size was controlled by varying the number of pipetting cycles, and pluripotency and proliferation were assessed via alkaline phosphatase staining and flow cytometry. Our results revealed that a decrease in clump size corresponded to an increase in cell proliferation, while pluripotency maintenance was optimized under specific clump sizes. These results underscore the significance of clump size for stem cell quality, emphasizing the need for a balanced approach to maintain pluripotency while fostering proliferation in the cell expansion culture for iPSCs.

Identification of Distinct, Quantitative Pattern Classes from Emergent Tissue-Scale hiPSC Bioelectric Properties.

Bioelectric signals possess the ability to robustly control and manipulate patterning during embryogenesis and tissue-level regeneration. Endogenous local and global electric fields function as a spatial 'pre-pattern', controlling cell fates and tissue-scale anatomical boundaries; however, the mechanisms facilitating these robust multiscale outcomes are poorly characterized. Computational modeling addresses the need to predict in vitro patterning behavior and further elucidate the roles of cellular bioelectric signaling components in patterning outcomes. Here, we modified a previously designed image pattern recognition algorithm to distinguish unique spatial features of simulated non-excitable bioelectric patterns under distinct cell culture conditions. This algorithm was applied to comparisons between simulated patterns and experimental microscopy images of membrane potential (Vmem) across cultured human iPSC colonies. Furthermore, we extended the prediction to a novel co-culture condition in which cell sub-populations possessing different ionic fluxes were simulated; the defining spatial features were recapitulated in vitro with genetically modified colonies. These results collectively inform strategies for modeling multiscale spatial characteristics that emerge in multicellular systems, characterizing the molecular contributions to heterogeneity of membrane potential in non-excitable cells, and enabling downstream engineered bioelectrical tissue design.

Abstract 3115: Mechanics And Genomic Profiles Reveal Aging And Developmental Lineage Heterogeneities In Human Induced Pluripotent Stem Cell-derived Smooth Muscle Cells.

Effective stem cell therapies and disease modeling depend on understanding epigenetic factors of aging. Accurately modeling age-dependent phenotypes such as aortic, and smooth muscle cell stiffening, requires cell models that reflect donor age. Improved knowledge of how donor aging influences stem cell-derived smooth muscle cells is essential for precise disease modeling and creating targeted therapeutics. We hypothesize that traits of vascular aging are retained in human induced pluripotent stem cells (iPSCs) and manifest in differentiated smooth muscle cells (SMCs). We aim to compare iPSC stiffness and gene expression between young (under 40, n=5 lines) and old (over 60, n=4 lines) donors, and examine age-dependent differences in their iPSC-SMCs. We measured cell stiffness using atomic force microscopy (AFM), and analyzed bulk and single-cell RNA gene expression in iPSCs and iPSC-SMCs of neuroectoderm (NE) or paraxial mesoderm (PM) lineages, respectively. AFM revealed no significant age-related differences in iPSC colony stiffness. Bulk RNA expression showed high similarity (>94% Pearson correlation) between young and old donor iPSCs, with principal component analysis indicating low variability and no clear age separation. Single-cell stiffness measurements found old NE-SMCs significantly stiffer than young ones (p=0.032), with no marked difference in PM-SMCs between ages. Old and young NE-SMCs were stiffer than their respective PM-SMC counterparts (old, p=0.006; young, p=0.007). UMAP analysis of single-cell RNA expression displayed distinct groupings for old and young NE-SMCs, but not for PM-SMCs. Cluster analysis revealed three clusters predominantly of old NE-SMCs and one of young NE-SMCs. Our study reveals age-related stiffening in NE lineage iPSC-SMCs, but not in PM lineage or iPSCs, indicating that iPSCs preserve certain donor age-related traits that manifest as lineage-specific phenotypes in SMCs post-differentiation. RNA sequencing showed similar expression patterns in young and old iPSCs, but age-specific differences in NE-SMCs. The integration of AFM and RNA expression data sheds light on the emergence of age-dependent phenotypes in differentiated, sensitive cell types, which are not present in immature cells.

High-volume, label-free imaging for quantifying single-cell dynamics in induced pluripotent stem cell colonies.

To facilitate the characterization of unlabeled induced pluripotent stem cells (iPSCs) during culture and expansion, we developed an AI pipeline for nuclear segmentation and mitosis detection from phase contrast images of individual cells within iPSC colonies. The analysis uses a 2D convolutional neural network (U-Net) plus a 3D U-Net applied on time lapse images to detect and segment nuclei, mitotic events, and daughter nuclei to enable tracking of large numbers of individual cells over long times in culture. The analysis uses fluorescence data to train models for segmenting nuclei in phase contrast images. The use of classical image processing routines to segment fluorescent nuclei precludes the need for manual annotation. We optimize and evaluate the accuracy of automated annotation to assure the reliability of the training. The model is generalizable in that it performs well on different datasets with an average F1 score of 0.94, on cells at different densities, and on cells from different pluripotent cell lines. The method allows us to assess, in a non-invasive manner, rates of mitosis and cell division which serve as indicators of cell state and cell health. We assess these parameters in up to hundreds of thousands of cells in culture for more than 36 hours, at different locations in the colonies, and as a function of excitation light exposure.

Molecular characterization of an iPSC line derived from a patient with cone‐rod dystrophy related to a mutation in the PROM1 gene

Aims/Purpose: The PROM1 gene encodes the protein Prominin‐1, which plays a crucial role in the morphogenesis of photoreceptor outer segments. The c.1354dupT mutation, associated with various inherited retinal diseases (IRD) phenotypes, leads to a premature stop codon. This study aimed to assess the silencing efficiency of reprogramming vectors and the expression of endogenous pluripotency genes in the iPSC line [DCB]‐FiPSC1‐Ep5F‐2.Methods: The iPSC line [DCB]‐FiPSC1‐Ep5F‐2 was generated from a patient with Cone‐rod dystrophy (CRD) linked to the c.1354dupT mutation in the PROM1 gene. iPSC colonies were stained using alkaline phosphatase (AP) blue membrane substrate. The c.1354dupT mutation was Sanger sequenced. To confirm the absence of episomal vector integration, quantitative PCR (qPCR) targeting a region common to all reprogramming vectors (within the EBNA1 gene) was conducted. Vector‐specific primer‐based quantitative reverse transcription PCR (qRT‐PCR) was used to assess the expression of reprogramming genes from the episomal vectors. Additionally, qRT‐PCR with gene‐specific primers was utilized to evaluate the expression of endogenous pluripotency genes. Untransfected fibroblasts served as the negative control, while fibroblasts transfected with the episomal vectors 72 h earlier were used as the positive control.Results: The iPSC line [DCB]‐FiPSC1‐Ep5F‐2 exhibited positive AP activity. The presence of the c.1354dupT mutation was confirmed. Furthermore, the number of copies of the EBNA1 gene was significantly lower in the generated iPSC line compared to the control. The relative mRNA expression of the reprogramming vectors was also significantly lower in the iPSC line, while the relative mRNA expression of the endogenous pluripotency genes was significantly higher than in the control.Conclusions: While further immunocytochemical characterization is necessary to fully ascertain iPSC pluripotency, the patient‐derived iPSC line [DCB]‐FiPSC1‐Ep5F‐2 shows promise as a disease model for personalized therapy development.

Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening.

C9orf72 genetic mutation is the most common genetic cause of ALS/FTD accompanied by abnormal protein insufficiency. Induced pluripotent stem cell (iPSC)-derived two-dimensional (2D) and three-dimensional (3D) cultures are providing new approaches. Therefore, this study established neuronal cell types and generated spinal cord organoids (SCOs) derived from C9orf72 knockdown human iPSCs to model ALS disease and screen the unrevealed phenotype. Wild-type (WT) iPSC lines from three healthy donor fibroblasts were established, and pluripotency and differentiation ability were identified by RT-PCR, immunofluorescence and flow cytometry. After infection by the lentivirus with C9orf72-targeting shRNA, stable C9-knockdown iPSC colonies were selected and differentiated into astrocytes, motor neurons and SCOs. Finally, we analyzed the extracted RNA-seq data of human C9 mutant/knockout iPSC-derived motor neurons and astrocytes from the GEO database and the inflammatory regulation-related genes in function and pathways. The expression of inflammatory factors was measured by qRT-PCR. The results showed that both WT-iPSCs and edited C9-iPSCs maintained a similar ability to differentiate into the three germ layers, astrocytes and motor neurons, forming SCOs in a 3D culture system. The constructed C9-SCOs have features of spinal cord development and multiple neuronal cell types, including sensory neurons, motor neurons, and other neurons. Based on the bioinformatics analysis, proinflammatory factors were confirmed to be upregulated in C9-iPSC-derived 2D cells and 3D cultured SCOs. The above differentiated models exhibited low C9orf72 expression and the pathological characteristics of ALS, especially neuroinflammation.

Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor T Cells: The Intersection of Stem Cells and Immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy is a promising cell-based immunotherapy applicable to various cancers. High cost of production, immune rejection, heterogeneity of cell product, limited cell source, limited expandability, and relatively long production time have created the need to achieve a universal allogeneic CAR-T cell product for "off-the-shelf" application. Since the innovation of induced pluripotent stem cells (iPSCs) by Yamanaka et al., extensive efforts have been made to prepare an unlimited cell source for regenerative medicine, that is, immunotherapy. In the autologous grafting approach, iPSCs prepare the desired cell source for generating autologous CAR-T cells through more accessible and available sources. In addition, generating iPSC-derived CAR-T cells is a promising approach to achieving a suitable source for producing an allogeneic CAR-T cell product. In brief, the first step is reprogramming somatic cells (accessible from peripheral blood, skin, etc.) to iPSCs. In the next step, CAR expression and T cell lineage differentiation should be applied in different arrangements. In addition, in an allogeneic manner, human leukocyte antigen/T cell receptor (TCR) deficiency should be applied in iPSC colonies. The allogeneic iPSC-derived CAR-T cell experiments showed that simultaneous performance of HLA/TCR deficiency, CAR expression, and T cell lineage differentiation could bring the production to the highest efficacy in generating allogeneic iPSC-derived CAR-T cells.

Size control of induced pluripotent stem cells colonies in two-dimensional culture for differentiation into functional monocyte-like cells

Background aimsMonocytes, derived from hematopoietic stem cells (HSCs), play a pivotal role in the immune response to cancer. Although they are an attractive source of cell therapy for cancer, a method for ex vivo expansion has not yet been established. Monocytes differentiated from pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs), can be an alternative source of HSC-derived monocytes because of their self-renewal and pluripotency. To develop a standardized method for the generation of iPSC-derived monocytes for future clinical applications, we aim to control the size of the iPSC colony. MethodsTo this end, we developed a plate with multiple dots containing a chemical substrate for the iPSC scaffold. iPSCs placed in the plate expanded only on the dots and created colonies of the same size. The cells were then differentiated into monocytes by adding cytokines to the colonies. ResultsThe dot plate substantially reduced variability in monocyte-like cell generation when compared with cultivating cells on a plate with the substrate covering the entire surface area. Furthermore, more monocyte-like cells were obtained by adjusting the dot size and the distance between the dots. The iPSC-derived monocyte-like cells phagocytosed cancer cells and secreted proinflammatory cytokines. The cells also expressed Fc receptors and exerted immunoglobulin G-mediated killing of cancer cells with the corresponding antibodies. ConclusionsThe dot plate enabled the control of iPSC colony size in two-dimensional culture, which resulted in a reduction in the generation-variation of functional monocyte-like cells. This standardized method for generating iPSC-derived monocyte-like cells using the dot plate could also facilitate the development of an automated closed system on a large scale for clinical applications.

Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells.

Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

YAP1 is essential for self-organized differentiation of pluripotent stem cells

Induced pluripotent stem cells (iPSCs) form aggregates that recapitulate aspects of the self-organization in early embryogenesis. Within few days, cells undergo a transition from epithelial-like structures to organized three-dimensional embryoid bodies (EBs) with upregulation of germ layer-specific genes. However, it is largely unclear, which signaling cascades regulate self-organized differentiation. The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. YAP1 has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human iPSCs remains to be elucidated. To gain insights into the function of YAP1 in early cell-fate decisions, we generated YAP1 knockout (YAP−/−) iPSC lines with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. YAP−/− iPSCs showed increased expression of several YAP1 targets and of NODAL, an important regulator of cell differentiation. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies towards endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP−/− lines. Notably, in undirected self-organized YAP−/− EBs germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the undirected self-organization of EBs, and this is at least partly attributed to activation of the NODAL signaling.

Attention-effective multiple instance learning on weakly stem cell colony segmentation

The detection of induced pluripotent stem cell (iPSC) colonies often needs the precise extraction of the colony features. However, existing computerized systems relied on segmentation of contours by preprocessing for classifying the colony conditions were task-extensive. To maximize the efficiency in categorizing colony conditions, we propose a multiple instance learning (MIL) in weakly supervised settings. It is designed in a single model to produce weak segmentation and classification of colonies without using finely labeled samples. As a single model, we employ a U-net-like convolution neural network (CNN) to train on binary image-level labels for MIL colonies classification. Furthermore, to specify the object of interest we used a simple post-processing method. The proposed approach is compared over conventional methods using five-fold cross-validation and receiver operating characteristic (ROC) curve. The maximum accuracy of the MIL-net is 95%, which is 15% higher than the conventional methods. Furthermore, the ability to interpret the location of the iPSC colonies based on the image level label without using a pixel-wise ground truth image is more appealing and cost-effective in colony condition recognition.

Generation of Induced Pluripotent Stem Cells from Lymphoblastoid Cell Lines by Electroporation of Episomal Vectors.

Lymphoblastoid cell lines (LCLs) deposited from disease-affected individuals could be a valuable donor cell source for generating disease-specific induced pluripotent stem cells (iPSCs). However, generation of iPSCs from the LCLs is still challenging, as yet no effective gene delivery strategy has been developed. Here, we reveal an effective gene delivery method specifically for LCLs. We found that LCLs appear to be refractory toward retroviral and lentiviral transduction. Consequently, lentiviral and retroviral transduction of OCT4, SOX2, KFL4 and c-MYC into LCLs does not elicit iPSC colony formation. Interestingly, however we found that transfection of oriP/EBNA-1-based episomal vectors by electroporation is an efficient gene delivery system into LCLs, enabling iPSC generation from LCLs. These iPSCs expressed pluripotency makers (OCT4, NANOG, SSEA4, SALL4) and could form embryoid bodies. Our data show that electroporation is an effective gene delivery method with which LCLs can be efficiently reprogrammed into iPSCs.

Characterizing the relationship between telomere length and DNA methylation age in induced pluripotent stem cells during neuronal differentiation

AbstractBackgroundInduced pluripotent stem cells (iPSCs) and their resultant neurons are popular models for studying diseases of aging, such as Alzheimer’s disease (AD). One hallmark of aging is the decreasing length of telomeres, the repetitive, protective DNA sequences at the end of each chromosome. We have previously demonstrated that whilst epigenetic age does increase with differentiation, iPSC‐derived neurons still have a fetal DNA methylation profile. It has also been shown by others that telomere length in iPSC‐derived neurons is shorter than the that of the stem cells from which they are derived. However, to our knowledge no one has looked at both epigenetic age and telomere length in the same samples. Therefore, we have collected DNA from different stages of the differentiation of iPSCs into neurons in order to look at the relationship between epigenetic age and telomere length.MethodHuman fibroblast cells were reprogrammed using lentiviral transfection. The resulting iPSC colonies were expanded, differentiated and collected at four different time points: day0 (iPSCs), day16 (neuronal progenitor cells), day37 and day58 (mature neurons). DNA methylation was assessed using the Illumina EPIC array and epigenetic age was derived using both the Horvath skin and blood clock and the Steg fetal brain clock. Absolute telomere length was determined using quantitative real‐time PCR.ResultThis study has identified the telomere dynamics of iPSCs as they undergo differentiation into cortical neurons. Not only this, but by also calculating the epigenetic age in the same samples we have been able to look at the relationship between telomere length and epigenetic age using two differentiation DNA methylation age calculators.ConclusionWe have outlined the relationship between telomere length and DNA methylation age of iPSCs as they differentiate into cortical neurons. This information is important to consider when using iPSCs to model age‐related diseases such as AD.

Highly efficient reprogrammable mouse lines with integrated reporters to track the route to pluripotency

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2°cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2°mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.

Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient.

We aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE) cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to provide potential cell sources for both basic scientific research and clinical application. In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR), karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCs exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and flow cytometry. The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated 99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong differentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC- RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1, Bestrophin, and PMEL17). A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with native RPE characteristics, which might provide potential regenerative treatments for AMD patients.

Combined Noncoding RNA-mRNA Regulomics Signature in Reprogramming and Pluripotency in iPSCs

Somatic cells are reprogrammed with reprogramming factors to generate induced pluripotent stem cells (iPSCs), offering a promising future for disease modeling and treatment by overcoming the limitations of embryonic stem cells. However, this process remains inefficient since only a small percentage of transfected cells can undergo full reprogramming. Introducing miRNAs, such as miR-294 and miR302/3667, with reprogramming factors, has shown to increase iPSC colony formation. Previously, we identified five transcription factors, GBX2, NANOGP8, SP8, PEG3, and ZIC1, which may boost iPSC generation. In this study, we performed quantitative miRNAome and small RNA-seq sequencing and applied our previously identified transcriptome to identify the potential miRNA-mRNA regulomics and regulatory network of other ncRNAs. From each fibroblast (N = 4), three iPSC clones were examined (N = 12). iPSCs and original fibroblasts expressed miRNA clusters differently and miRNA clusters were compared to mRNA hits. Moreover, miRNA, piRNA, and snoRNAs expression profiles in iPSCs and original fibroblasts were assessed to identify the potential role of ncRNAs in enhancing iPSC generation, pluripotency, and differentiation. Decreased levels of let-7a-5p showed an increase of SP8 as described previously. Remarkably, the targets of identifier miRNAs were grouped into pluripotency canonical pathways, on stemness, cellular development, growth and proliferation, cellular assembly, and organization of iPSCs.

Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3–4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

Efficient reprogramming of human fibroblasts using RNA reprogramming with DAPT and iDOT1L under normoxia conditions.

IntroductionHuman induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells expressing a defined set of transcription factors. The advent of autologous iPSCs has enabled the generation of patient-specific iPSC lines and is expected to contribute to the exploration of cures and causes of diseases, drug screening, and tailor-made regenerative medicines. Efficient control of hiPSC derivation is beneficial for industrial applications. However, the mechanisms underlying somatic cell reprogramming remain unknown, while reprogramming efficiency remains extremely low, especially in human cells.Methods and resultsWe previously reported that chemical inhibition of the NOTCH signaling pathway and DOT1L promoted the generation of hiPSCs from keratinocytes, but the mechanisms and effect of this double inhibition on other types of cells remain to be investigated. Here, we found that the NOTCH/DOT1L inhibition markedly increased iPSC colony generation from human fibroblast cells via mRNA reprogramming, and mesenchymal to epithelial transition (MET)-related genes are significantly expressed in the early phase of the reprogramming. We successfully derived hiPSC lines using a single-cell sorting system under efficient reprogramming conditions.ConclusionsThis user-friendly reprogramming approach paves the way for the development of hiPSC derivations in industrial applications of disease modeling and drug screening.