Thiamine-modified metabolic reprogramming of human pluripotent stem cell-derived cardiomyocyte under space microgravity.

During spaceflight, the cardiovascular system undergoes remarkable adaptation to microgravity and faces the risk of cardiac remodeling. Therefore, the effects and mechanisms of microgravity on cardiac morphology, physiology, metabolism, and cellular biology need to be further investigated. Since China started constructing the China Space Station (CSS) in 2021, we have taken advantage of the Shenzhou-13 capsule to send human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to the Tianhe core module of the CSS. In this study, hPSC-CMs subjected to space microgravity showed decreased beating rate and abnormal intracellular calcium cycling. Metabolomic and transcriptomic analyses revealed a battery of metabolic remodeling of hPSC-CMs in spaceflight, especially thiamine metabolism. The microgravity condition blocked the thiamine intake in hPSC-CMs. The decline of thiamine utilization under microgravity or by its antagonistic analog amprolium affected the process of the tricarboxylic acid cycle. It decreased ATP production, which led to cytoskeletal remodeling and calcium homeostasis imbalance in hPSC-CMs. More importantly, in vitro and in vivo studies suggest that thiamine supplementation could reverse the adaptive changes induced by simulated microgravity. This study represents the first astrobiological study on the China Space Station and lays a solid foundation for further aerospace biomedical research. These data indicate that intervention of thiamine-modified metabolic reprogramming in human cardiomyocytes during spaceflight might be a feasible countermeasure against microgravity.

Simple modeling of familial Alzheimer's disease using human pluripotent stem cell-derived cerebral organoid technology.

BACKGROUND: Cerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer's disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening. METHODS: We established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-ß (Aß) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky's staining, and western blot analysis. RESULTS: The AD COs exhibited extensive Aß accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a ß-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features. CONCLUSION: Our model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation.

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

The Role of Human Pluripotent Stem Cells in Amyotrophic Lateral Sclerosis: From Biological Mechanism to Practical Implications.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, characterized by progressive loss of both upper and lower motor neurons, resulting in clinical features such as muscle weakness, paralysis, and ultimately, respiratory failure. Nowadays, there is not effective treatment to reverse the progression of the disease, that leads to death within 3-5 years after the onset. Nevertheless, the induced pluripotent stem cells (iPS) technology could be the answer, providing disease modelling, drug testing, and cell-based therapies for this pathology. The aim of this work was to conduct a literature review of the past 5 years about the role of iPS in ALS, to better define the neurobiological mechanisms involved in the pathogenesis and the potential future therapies. The review also deals with advanced and currently available technologies used to reprogram cell lines and generate human motor neurons in vitro, which represent the source to study the pathological processes, the relationship between phenotype and genotype, the disease progression and the potential therapeutic targets of these group of disorders. Specific treatment options with stem cells involve Advance Gene Editing Technology, neuroprotective agents, and cells or exosomes transplantation, aimed to replace dead or damaged nerve cells. In summary, this review comprehensively addresses the role of human pluripotent stem cells (hPSCs) in motor neuron diseases (MND), with a focus on physiopathology, diagnostic and prognostic implications, specific and potential future treatment options. Understanding the biological mechanisms and practical implications of hPSCs in MND is crucial for advancing therapeutic strategies and improving outcomes for patients affected by these devastating diseases.

SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss.

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, because cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but their functional consequences in differentiating cells are scarcely described. In this work, we show that the loss of chromosome 18q impairs neuroectoderm commitment and that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for this failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, and transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, suggesting that these cells are in an altered state of pluripotency.

The role of lipids in genome integrity and pluripotency.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research.

Direct programming of human pluripotent stem cells into endothelial progenitors with SOX17 and FGF2.

Transcription factors (TFs) are pivotal in guiding stem cell behavior, including their maintenance and differentiation. Using single-cell RNA sequencing, we investigated TFs expressed in endothelial progenitors (EPs) derived from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Conversely, SOX17 knockdown via CRISPR-Cas13d significantly compromised EP differentiation. Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31- cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into endothelial cells. These findings underscore an undiscovered role of SOX17 in programming hPSCs toward an EP lineage, illuminating pivotal mechanisms in EP differentiation.

The Epiblast and Pluripotent Stem Cell Lines.

All somatic cells develop from the epiblast, which occupies the upper layer of two-layered embryos and in most mammals is formed after the implantation stage but before gastrulation initiates. Once the epiblast is established, the epiblast cells begin to develop into various somatic cells via large-scale cell reorganization, namely, gastrulation. Different pluripotent stem cell lines representing distinct stages of embryogenesis have been established: mouse embryonic stem cells (mESCs), human embryonic stem cells (hESCs), and mouse epiblast stem cells (EpiSCs), which represent the preimplantation stage inner cell mass, an early  post-implantation stage epiblast, and a later-stage epiblast, respectively. Together, these cell lines provide excellent in vitro models of cell regulation before somatic cells develop. This chapter addresses these early developmental stages.

MiR-290 Family Maintains Pluripotency and Self-Renewal by Regulating MAPK Signaling Pathway in Intermediate Pluripotent Stem Cells.

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are derived from pre- and post-implantation embryos, representing the initial "naïve" and final "primed" states of pluripotency, respectively. In this study, novel reprogrammed pluripotent stem cells (rPSCs) were induced from mouse EpiSCs using a chemically defined medium containing mouse LIF, BMP4, CHIR99021, XAV939, and SB203580. The rPSCs exhibited domed clones and expressed key pluripotency genes, with both X chromosomes active in female cells. Furthermore, rPSCs differentiated into cells of all three germ layers in vivo through teratoma formation. Regarding epigenetic modifications, the DNA methylation of Oct4, Sox2, and Nanog promoter regions and the mRNA levels of Dnmt3a, Dnmt3b, and Dnmt1 were reduced in rPSCs compared with EpiSCs. However, the miR-290 family was significantly upregulated in rPSCs. After removing SB203580, an inhibitor of the p38 MAPK pathway, the cell colonies changed from domed to flat, with a significant decrease in the expression of pluripotency genes and the miR-290 family. Conversely, overexpression of pri-miR-290 reversed these changes. In addition, Map2k6 was identified as a direct target gene of miR-291b-3p, indicating that the miR-290 family maintains pluripotency and self-renewal in rPSCs by regulating the MAPK signaling pathway.

DPPA2/4 Promote the Pluripotency and Proliferation of Bovine Extended Pluripotent Stem Cells by Upregulating the PI3K/AKT/GSK3ß/ß-Catenin Signaling Pathway.

Developmental pluripotency-associated 2 (DPPA2) and DPPA4 are crucial transcription factors involved in maintaining pluripotency in humans and mice. However, the role of DPPA2/4 in bovine extended pluripotent stem cells (bEPSCs) has not been investigated. In this study, a subset of bEPSC-related differentially expressed genes (DEGs), including DPPA2 and DPPA4, was identified based on multiomics data (ATAC-seq and RNA-seq). Subsequent investigations revealed that double overexpression of DPPA2/4 facilitates the reprogramming of bovine fetal fibroblasts (BFFs) into bEPSCs, whereas knockout of DPPA2/4 in BFFs leads to inefficient reprogramming. DPPA2/4 overexpression and knockdown experiments revealed that the pluripotency and proliferation capability of bEPSCs were maintained by promoting the transition from the G1 phase to the S phase of the cell cycle. By activating the PI3K/AKT/GSK3ß/ß-catenin pathway in bEPSCs, DPPA2/4 can increase the nuclear accumulation of ß-catenin, which further upregulates lymphoid enhancer binding factor 1 (LEF1) transcription factor activity. Moreover, DPPA2/4 can also regulate the expression of LEF1 by directly binding to its promoter region. Overall, our results demonstrate that DPPA2/4 promote the reprogramming of BFFs into bEPSCs while also maintaining the pluripotency and proliferation capability of bEPSCs by regulating the PI3K/AKT/GSK3ß/ß-catenin pathway and subsequently activating LEF1. These findings expand our understanding of the gene regulatory network involved in bEPSC pluripotency.

Determining the potency of primordial germ cells by injection into early mouse embryos.

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution.

Bovine Pluripotent Stem Cells: Current Status and Prospects.

Pluripotent stem cells (PSCs) can differentiate into three germ layers and diverse autologous cell lines. Since cattle are the most commonly used large domesticated animals, an important food source, and bioreactors, great efforts have been made to establish bovine PSCs (bPSCs). bPSCs have great potential in bovine breeding and reproduction, modeling in vitro differentiation, imitating cancer development, and modeling diseases. Currently, bPSCs mainly include bovine embryonic stem cells (bESCs), bovine induced pluripotent stem cells (biPSCs), and bovine expanded potential stem cells (bEPSCs). Establishing stable bPSCs in vitro is a critical scientific challenge, and researchers have made numerous efforts to this end. In this review, the category of PSC pluripotency; the establishment of bESCs, biPSCs, and bEPSCs and its challenges; and the application outlook of bPSCs are discussed, aiming to provide references for future research.

Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galß1-3GlcNAcß1-3Galß1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galß1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Unsupervised analysis of whole transcriptome data from human pluripotent stem cells cardiac differentiation.

The main objective of the present work was to highlight differences and similarities in gene expression patterns between different pluripotent stem cell cardiac differentiation protocols, using a workflow based on unsupervised machine learning algorithms to analyse the transcriptome of cells cultured as a 2D monolayer or as 3D aggregates. This unsupervised approach effectively allowed to portray the transcriptomic changes that occurred throughout the differentiation processes, with a visual representation of the entire transcriptome. The results allowed to corroborate previously reported data and also to unveil new gene expression patterns. In particular, it was possible to identify a correlation between low cardiomyocyte differentiation efficiencies and the early expression of a set of non-mesodermal genes, which can be further explored as predictive markers of differentiation efficiency. The workflow here developed can also be applied to analyse other stem cell differentiation transcriptomic datasets, envisaging future clinical implementation of cellular therapies.

Identification of specific markers for human pluripotent stem cell-derived small extracellular vesicles.

Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs.

POZ/BTB and AT hook containing zinc finger 1 (PATZ1) suppresses differentiation and regulates metabolism in human embryonic stem cells.

Human embryonic stem cells (hESCs) can proliferate infinitely (self-renewal) and give rise to almost all types of somatic cells (pluripotency). Hence, understanding the molecular mechanism of pluripotency regulation is important for applications of hESCs in regenerative medicine. Here we report that PATZ1 is a key factor that regulates pluripotency and metabolism in hESCs. We found that depletion of PATZ1 is associated with rapid downregulation of master pluripotency genes and prominent deceleration of cell growth. We also revealed that PATZ1 regulates hESC pluripotency though binding the regulatory regions of OCT4 and NANOG. In addition, we demonstrated PATZ1 is a key node in the OCT4/NANOG transcriptional network. We further revealed that PATZ1 is essential for cell growth in hESCs. Importantly, we discovered that depletion of PATZ1 drives hESCs to exploit glycolysis which energetically compensates for the mitochondrial dysfunction. Overall, our study establishes the fundamental role of PATZ1 in regulating pluripotency in hESCs. Moreover, PATZ1 is essential for maintaining a steady metabolic homeostasis to refine the stemness of hESCs.

Dynamic nucleolar phase separation influenced by non-canonical function of LIN28A instructs pluripotent stem cell fate decisions.

LIN28A is important in somatic reprogramming and pluripotency regulation. Although previous studies addressed that LIN28A can repress let-7 microRNA maturation in the cytoplasm, few focused on its role within the nucleus. Here, we show that the nucleolus-localized LIN28A protein undergoes liquid-liquid phase separation (LLPS) in mouse embryonic stem cells (mESCs) and in vitro. The RNA binding domains (RBD) and intrinsically disordered regions (IDR) of LIN28A contribute to LIN28A and the other nucleolar proteins' phase-separated condensate establishment. S120A, S200A and R192G mutations in the IDR result in subcellular mislocalization of LIN28A and abnormal nucleolar phase separation. Moreover, we find that the naive-to-primed pluripotency state conversion and the reprogramming are associated with dynamic nucleolar remodeling, which depends on LIN28A's phase separation capacity, because the LIN28A IDR point mutations abolish its role in regulating nucleolus and in these cell fate decision processes, and an exogenous IDR rescues it. These findings shed light on the nucleolar function in pluripotent stem cell states and on a non-canonical RNA-independent role of LIN28A in phase separation and cell fate decisions.

PRAMEL7 and CUL2 decrease NuRD stability to establish ground-state pluripotency.

Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency.

Notch3 directs differentiation of brain mural cells from human pluripotent stem cell-derived neural crest.

Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3. Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRß, TBX2, FOXS1, KCNJ8, SLC6A12, and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional KATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.

Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.