Detection of senescent cells in the mucosal healing process on type 2 diabetic rats after tooth extraction for biomaterial development.

The delayed mucosal healing of tooth extraction sockets in diabetes has few known effective treatment strategies, and its underlying mechanism remains unknown. Senescent cells may play a pivotal role in this delay, given the well-established association between diabetes, senescent cells, and wound healing. Here, we demonstrated an increase in p21- or p16-positive senescent cells in the epithelial and connective tissues of extraction sockets in type 2 diabetic rats compared to those in control rats. Between 7 and 14 days after tooth extraction, a decrease in senescent cells and improvement in re-epithelialization failure were observed in the epithelium, while an increase in senescent cells and persistence of inflammation were observed in the connective tissue. These results suggest that cellular senescence may have been induced by diabetes and contributed to delayed mucosal healing by suppressing re-epithelization and persistent inflammation. These findings provide new targets for treatment using biomaterials, cells, and drugs.

Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs.

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.

Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.

Generation of chimpanzee induced pluripotent stem cell lines for cross-species comparisons.

As humans' closest living relatives, chimpanzees offer valuable insights into human evolution. However, technical and ethical limitations hinder investigations into the molecular and cellular foundations that distinguish chimpanzee and human traits. Recently, induced pluripotent stem cells (iPSCs) have emerged as a novel model for functional comparative studies and provided a non-invasive alternative for studying embryonic phenomena. In this study, we generated five new chimpanzee iPSC lines from peripheral blood cells and skin fibroblasts with SeV vectors carrying four reprogramming factors (human OCT3/4, SOX2, KLF4, and L-MYC) and characterized their pluripotency and differentiation potential. We also examined the expression of a human-specific non-coding RNA, HSTR1, which is predicted to be involved in human brain development. Our results show that the chimpanzee iPSCs possess pluripotent characteristics and can differentiate into various cell lineages. Moreover, we found that HSTR1 is expressed in human iPSCs and their neural derivatives but not in chimpanzee counterparts, supporting its possible role in human-specific brain development. As iPSCs are inherently variable due to genetic and epigenetic differences in donor cells or reprogramming procedures, it is essential to expand the number of chimpanzee iPSC lines to comprehensively capture the molecular and cellular properties representative of chimpanzees. Hence, our cells provide a valuable resource for investigating the function and regulation of human-specific transcripts such as HSTR1 and for understanding human evolution more generally.

Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells.

Introduction: Basic fibroblast growth factor (bFGF, FGF2) and integrin α6ß1 are important for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Although bFGF-integrin binding contributes to biofunctions in cancer cells, the relationship in hPSCs remains unclear. Methods: To investigate the relationship between bFGF and integrin in human induced pluripotent stem cells (hiPSCs), we generated recombinant human bFGF wild-type and mutant proteins, that do not bind to integrin, FGFR, or both. We then cultured hiPSCs with these recombinant bFGF proteins. To evaluate the abilities of recombinant bFGF proteins in maintaining hPSC properties, pluripotent markers, ERK activity, and focal adhesion structure were analyzed through flow cytometry, immunofluorescence (IF), and immunoblotting (IB). Result: We identified an interaction between bFGF and integrin α6ß1 in vitro and in hiPSCs. The integrin non-binding mutant was incapable of inducing the hPSC properties, such as proliferation, ERK activity, and large focal adhesions at the edges of hiPSC colonies. Signaling induced by bFGF-FGFR binding was essential during the first 24 h after cell seeding for maintaining the properties of hPSCs, followed by a shift towards intracellular signaling via the bFGF-integrin interaction. The mixture of the two bFGF mutants also failed to maintain hPSC properties, indicating that bFGF binds to both FGFR and integrin. Conclusion: Our study demonstrates that the integrin-bFGF-FGFR ternary complex maintains the properties of hPSCs via intracellular signaling, providing insights into the functional crosstalk between bFGF and integrins in hiPSCs.

Culturing human pluripotent stem cells for regenerative medicine.

INTRODUCTION: The development of human pluripotent stem cell (hPSC) culture protocols has led to the establishment of directed differentiation induction methods, resulting in their application in regenerative medicine. Cell therapy products derived from hPSCs have been transplanted into patients, and promising results have been observed in some ongoing clinical trials. AREA COVERED: This review provides an overview of the challenges associated with the culture of hPSCs for clinical applications and the development of culture technologies designed to address these challenges. We also review future cell culture strategies for large-scale manufacturing to enhance patient access. EXPERT OPINION: Despite the great potential of hPSCs, difficulties such as safety, quality control, and cost management continue to pose obstacles to their product development and clinical translation. A substantial contribution of these issues lies in the cell culture process. Therefore, selecting the appropriate ancillary materials (AMs) and integrating effective culture methods in standard operating procedures (SOPs) from the early stages of clinical development are essential for success. Moreover, incorporating an automated scaling process is imperative to ensure the commercial feasibility of hPSC-based products. [Figure: see text]Human pluripotent stem cells (hPSCs) show great potential as a valuable resource for regenerative medicine. However, three significant obstacles must be overcome: safety, quality, and costs. Thankfully, recent progress in hPSC culture techniques has effectively tackled these challenges, opening up exciting possibilities for realizing hPSC-based regenerative medicine.

Localization of senescent cells under cavity preparations in rats and restoration of reparative dentin formation by senolytics.

Reparative dentin formed by dental cavity preparation (DCP) is frequently used in clinical operations and plays a pivotal role in pulp protection. Recent reports have shown that senescent cells induced by various stressors aggravate many diseases. They can be treated using senolytics, which are drugs that selectively eliminate senescent cells. However, the association between DCP, senescent cells, and senolytics remains unclear. In this study, we established a rat model of DCP and analyzed the spatiotemporal localization of senescent cells in the pulp. The results showed that p21- and p16-positive senescent cells appeared mostly around the pulp horn (PH) under DCP. Furthermore, administration of senolytics (dasatinib and quercetin) successfully eliminated these senescent cells, thereby restoring the volume of reparative dentin formation. These data indicate that senescent cells induced by DCP may hamper the formation of reparative dentin. Senescent cells may be targets for the development of new restorative dentistry therapies.

Identification of Senescent Cells in Peri-Implantitis and Prevention of Mini-Implant Loss Using Senolytics.

Peri-implantitis is a disease that causes the detachment of orthodontic mini-implants. Recently, stress-induced senescent cells have been reported to be involved in various inflammatory diseases. Senescent cell-eliminating drugs, termed "senolytics", can improve the symptoms of such diseases. However, the relationship between peri-implantitis and senescent cells remains unclear. In this study, we evaluated the presence of senescent cells in a rat peri-implantitis model developed with a gum ring. The effect on bone resorption and implant loss was also investigated with and without senolytics (Dasatinib and Quercetin). The number of senescence markers (p19, p21, and p16) was found to increase, and implant detachment occurred in 24 days. After the administration of senolytics, the number of senescence markers decreased and implant detachment was inhibited. This study suggests that senescent cells aggravate peri-implantitis and senolytic administration latently reduces implant loss by inhibiting senescence-related mechanisms.

Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells.

The effects of transcription factors on the maintenance and differentiation of human-induced or embryonic pluripotent stem cells (iPSCs/ESCs) have been well studied. However, the importance of posttranscriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 posttranscriptionally regulated genes with strict upregulation of the protein level in iPSCs/ESCs. Among them, we found 84 genes were vital for the survival of iPSCs and HDFs, including 20 genes that were specifically necessary for iPSC survival. These 20 proteins were upregulated only in iPSCs/ESCs and not in differentiated cells derived from the three germ layers. Although there are still unknown mechanisms that downregulate protein levels in HDFs, these results reveal that posttranscriptionally regulated genes have a crucial role in iPSC survival.

MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains.

Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.

Inherent genomic properties underlie the epigenomic heterogeneity of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) show variable differentiation potential due to their epigenomic heterogeneity, whose extent/attributes remain unclear, except for well-studied elements/chromosomes such as imprints and the X chromosomes. Here, we show that seven hiPSC lines with variable germline potential exhibit substantial epigenomic heterogeneity, despite their uniform transcriptomes. Nearly a quarter of autosomal regions bear potentially differential chromatin modifications, with promoters/CpG islands for H3K27me3/H2AK119ub1 and evolutionarily young retrotransposons for H3K4me3. We identify 145 large autosomal blocks (≥100 kb) with differential H3K9me3 enrichment, many of which are lamina-associated domains (LADs) in somatic but not in embryonic stem cells. A majority of these epigenomic heterogeneities are independent of genetic variations. We identify an X chromosome state with chromosome-wide H3K9me3 that stably prevents X chromosome erosion. Importantly, the germline potential of female hiPSCs correlates with X chromosome inactivation. We propose that inherent genomic properties, including CpG density, transposons, and LADs, engender epigenomic heterogeneity in hiPSCs.

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin ß1-ILK-ß-catenin-JUN axis.

Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/ß-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin ß1 (ITGB1) and downstream integrin-linked protein kinase (ILK), ß-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/ß-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-ß-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general.

Removal of Interference MS/MS Spectra for Accurate Quantification in Isobaric Tag-Based Proteomics.

Rapid progress in mass spectrometry (MS) has made comprehensive analyses of the proteome possible, but accurate quantification remains challenging. Isobaric tags for relative and absolute quantification (iTRAQ) is widely used as a tool to quantify proteins expressed in different cell types and various cellular conditions. The quantification precision of iTRAQ is quite high, but the accuracy dramatically decreases in the presence of interference peptides that are coeluted and coisolated with the target peptide. Here, we developed "removal of interference mixture MS/MS spectra (RiMS)" to improve the quantification accuracy of isobaric tag approaches. The presence of spectrum interference is judged by examining the overlap in the elution time of all scanned precursor ions. Removal of this interference decreased protein identification (11% loss) but improved quantification accuracy. Further, RiMS does not require any specialized equipment, such as MS3 instruments or an additional ion separation mode. Finally, we demonstrated that RiMS can be used to quantitatively compare human-induced pluripotent stem cells and human dermal fibroblasts, as it revealed differential protein expressions that reflect the biological characteristics of the cells.

Human Pluripotent Stem Cell-Derived Tumor Model Uncovers the Embryonic Stem Cell Signature as a Key Driver in Atypical Teratoid/Rhabdoid Tumor.

Atypical teratoid/rhabdoid tumor (AT/RT), which harbors SMARCB1 mutation and exhibits a characteristic histology of rhabdoid cells, has a poor prognosis because of the lack of effective treatments. Here, we establish human SMARCB1-deficient pluripotent stem cells (hPSCs). SMARCB1-deficient hPSC-derived neural progenitor-like cells (NPLCs) efficiently give rise to brain tumors when transplanted into the mouse brain. Notably, activation of an embryonic stem cell (ESC)-like signature confers a rhabdoid histology in SMARCB1-deficient NPLC-derived tumors and causes a poor prognosis. Consistently, we find the activation of the ESC-like gene expression signature and an ESC-like DNA methylation landscape in clinical specimens of AT/RT. Finally, we identify candidate genes that maintain the activation of the ESC-like signature and the growth of AT/RT cells. Collectively, SMARCB1-deficient hPSCs offer the human models for AT/RT, which uncover the role of the activated ESC-like signature in the poor prognosis and unique histology of AT/RT.

Induced Pluripotent Stem Cells and Their Use in Human Models of Disease and Development.

The discovery of somatic cell nuclear transfer proved that somatic cells can carry the same genetic code as the zygote, and that activating parts of this code are sufficient to reprogram the cell to an early developmental state. The discovery of induced pluripotent stem cells (iPSCs) nearly half a century later provided a molecular mechanism for the reprogramming. The initial creation of iPSCs was accomplished by the ectopic expression of four specific genes (OCT4, KLF4, SOX2, and c-Myc; OSKM). iPSCs have since been acquired from a wide range of cell types and a wide range of species, suggesting a universal molecular mechanism. Furthermore, cells have been reprogrammed to iPSCs using a myriad of methods, although OSKM remains the gold standard. The sources for iPSCs are abundant compared with those for other pluripotent stem cells; thus the use of iPSCs to model the development of tissues, organs, and other systems of the body is increasing. iPSCs also, through the reprogramming of patient samples, are being used to model diseases. Moreover, in the 10 years since the first report, human iPSCs are already the basis for new cell therapies and drug discovery that have reached clinical application. In this review, we examine the generation of iPSCs and their application to disease and development.

Derivation of induced pluripotent stem cells in Japanese macaque (Macaca fuscata).

Non-human primates are our closest relatives and are of special interest for ecological, evolutionary and biomedical research. The Japanese macaque (Macaca fuscata) has contributed to the progress of primatology and neurosciences over 60 years. Despite this importance, the molecular and cellular basis of the Japanese macaque remains unexplored since useful cellular tools are lacking. Here we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of the Japanese macaque with Sendai virus or plasmid vectors. The Japanese macaque iPSCs (jm-iPSCs) were established under feeder-free culture conditions, but feeder cells turned out to be essential for their maintenance. The jm-iPSCs formed human iPSC-like flat colonies which were positive for pluripotent antigens including alkaline phosphatase, SSEA4, and TRA-1-81. They also expressed endogenous OCT3/4, SOX2, L-MYC, and KLF4 and other pluripotent marker genes. The potential to differentiate into all three germ layers and neural stem cells was confirmed by embryoid body and neurosphere formation, respectively. The jm-iPSCs will provide a robust in vitro tool for investigating the underlying mechanisms of development and physiology studies with the Japanese macaque.

Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Repurposing the Cord Blood Bank for Haplobanking of HLA-Homozygous iPSCs and Their Usefulness to Multiple Populations.

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three-germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)-based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA-homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA-homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks. Stem Cells 2018;36:1552-1566.

iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid ß Combination for Alzheimer's Disease.

In the process of drug development, in vitro studies do not always adequately predict human-specific drug responsiveness in clinical trials. Here, we applied the advantage of human iPSC-derived neurons, which offer human-specific drug responsiveness, to screen and evaluate therapeutic candidates for Alzheimer's disease (AD). Using AD patient neurons with nearly 100% purity from iPSCs, we established a robust and reproducible assay for amyloid ß peptide (Aß), a pathogenic molecule in AD, and screened a pharmaceutical compound library. We acquired 27 Aß-lowering screen hits, prioritized hits by chemical structure-based clustering, and selected 6 leading compounds. Next, to maximize the anti-Aß effect, we selected a synergistic combination of bromocriptine, cromolyn, and topiramate as an anti-Aß cocktail. Finally, using neurons from familial and sporadic AD patients, we found that the cocktail showed a significant and potent anti-Aß effect on patient cells. This human iPSC-based platform promises to be useful for AD drug development.