Stem cell challenges and opportunities.

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s)", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors.

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hypermethylated regions compared with embryonic stem cells. These random aberrant hypermethylations might be a cause of the differences in the properties of each of the iPSC lines.

DNA hypermethylation enhanced telomerase reverse transcriptase expression in human-induced pluripotent stem cells.

During reprogramming into human induced pluripotent stem cells (iPSCs), several stem cell marker genes are induced, such as OCT-4, NANOG, SALL4, and TERT. OCT-4, NANOG, and SALL4 gene expression can be regulated by DNA methylation. Their promoters become hypomethylated in iPSCs during reprogramming, leading to their induced expression. However, epigenetic regulation of the TERT gene remains unclear. In this study, we focused on epigenetic regulation of the human TERT gene and identified a differentially methylated region (DMR) at a distal region in the TERT promoter between human iPSCs and their parental somatic cells. Interestingly, the TERT-DMR was highly methylated in iPSCs, but low-level methylation was observed in their parental somatic cells. Region-specific, methylated-promoter assays showed that the methylated TERT-DMR up-regulated the promoter activity in iPSCs. In addition, Lamin B1 accumulated at the TERT-DMR in iPSCs, but not in their parent somatic cells. These results suggested that the TERT transcription was enhanced by DNA methylation at the TERT-DMR via binding to nuclear lamina during reprogramming. Our findings shed light on a new functional aspect of DNA methylation in gene expression.

Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders.

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications.

In vivo maturation of human embryonic stem cell-derived teratoma over time.

Transformation of human embryonic stem cells (hESC) is of interest to scientists who use them as a raw material for cell-processed therapeutic products. However, the WHO and ICH guidelines provide only study design advice and general principles for tumorigenicity tests. In this study, we performed in vivo tumorigenicity tests (teratoma formation) and genome-wide sequencing analysis of undifferentiated hESCs i.e. SEES-1, -2 and -3 cells. We followed up with teratoma formation histopathologically after subcutaneous injection of SEES cells into immunodeficient mice in a qualitative manner and investigated the transforming potential of the teratomas. Maturity of SEES-teratomas perceptibly increased after long-term implantation, while areas of each tissue component remained unchanged. We found neither atypical cells/structures nor cancer in the teratomas even after long-term implantation. The teratomas generated by SEES cells matured histologically over time and did not increase in size. We also analyzed genomic structures and sequences of SEES cells during cultivation by SNP bead arrays and next-generation sequencing, respectively. The nucleotide substitution rate was 3.1 × 10-9, 4.0 × 10-9, and 4.6 × 10-9 per each division in SEES-1, SEES-2, and SEES-3 cells, respectively. Heterozygous single-nucleotide variations were detected, but no significant homologous mutations were found. Taken together, these results imply that SEES-1, -2, and -3 cells do not exhibit in vivo transformation and in vitro genomic instability.

Glycolipid dynamics in generation and differentiation of induced pluripotent stem cells.

Glycosphingolipids (GSLs) are glycoconjugates that function as mediators of cell adhesion and modulators of signal transduction. Some well-defined markers of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are glycoconjugates, such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. However, Comprehensive GSL profiles of hiPSCs have not yet been elucidated. The global images of GSLs from the parental cells, hiPSCs, and differentiated cells revealed that there are parental cell-independent specific glycolipids, including Globo H (fucosyl-Gb5Cer) and H type1 antigen (fucosyl-Lc4Cer) that are novel markers for undifferentiated hiPSCs. Interestingly, undifferentiated hiPSCs expressed H type 1 antigen, specific for blood type O, regardless of the cells' genotypes. Thus, in this study, we defined the dynamics of GSL remodeling during reprogramming from parental cell sets to iPSC sets and thence to iPSC-neural cells.

Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1, -2, -3, -4, -5, -6, and -7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome.

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and ß cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.

Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability.

Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a comparable nucleotide substitution rate in AT-iPS cells. Taken together, these data show that ATM is involved in protection from irradiation-induced cell death.

Large-scale cell production of stem cells for clinical application using the automated cell processing machine.

BACKGROUND: Cell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, we focused on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported till date. The system AutoCulture used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. We examined the feasibility of this system in a clinical setting. RESULTS: We observed similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture is a feasible method for the cultivation of human cells for regenerative medicine. CONCLUSIONS: An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products.

Glycan profiling of endometrial cancers using lectin microarray.

Cell surface glycans change during the process of malignant transformation. To characterize and distinguish endometrial cancer and endometrium, we performed glycan profiling using an emerging modern technology, lectin microarray analysis. The three cell lines, two from endometrial cancers [well-differentiated type (G1) and poorly differentiated type (G3)] and one from normal endometrium, were successfully categorized into three independent groups by 45 lectins. Furthermore, in cancer cells, a clear difference between G1 and G3 type was observed for the glycans recognized with six lectins, Ulex europaeus agglutinin I (UEA-I), Sambucus sieboldiana agglutinin (SSA), Sambucus nigra agglutinin (SNA), Trichosanthes japonica agglutinin I (TJA-I), Amaranthus caudatus agglutinin (ACA), and Bauhinia purpurea lectin (BPL). The lectin microarray analysis using G3 type tissues demonstrated that stage I and stage III or IV were distinguished depending on signal pattern of three lectins, Dolichos biflorus agglutinin (DBA), BPL, and ACA. In addition, the analysis of the glycans on the ovarian cancer cells showed that only anticancer drug-sensitive cell lines had almost no activities to specific three lectins. Glycan profiling by the lectin microarray may be used to assess the characteristics of tumors and potentially to predict the success of chemotherapy treatment.

DNA methylation dynamics in human induced pluripotent stem cells over time.

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.

Lectin microarray analysis of pluripotent and multipotent stem cells.

Stem cells have a capability to self-renew and differentiate into multiple types of cells; specific markers are available to identify particular stem cells for developmental biology research. In this study, we aimed to define the status of somatic stem cells and the pluripotency of human embryonic stem (hES) and induced pluripotent stem (iPS) cells using a novel molecular methodology, lectin microarray analysis. Our lectin microarray analysis successfully categorized murine somatic stem cells into the appropriate groups of differentiation potency. We then classified hES and iPS cells by the same approach. Undifferentiated hES cells were clearly distinguished from differentiated hES cells after embryoid formation. The pair-wise comparison means based on 'false discovery rate' revealed that three lectins -Euonymus europaeus lectin (EEL), Maackia amurensis lectin (MAL) and Phaseolus vulgaris leucoagglutinin [PHA(L)]- generated maximal values to define undifferentiated and differentiated hES cells. Furthermore, to define a pluripotent stem cell state, we generated a discriminant for the undifferentiated state with pluripotency. The discriminant function based on lectin reactivities was highly accurate for judgment of stem cell pluripotency. These results suggest that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.

Defining hypo-methylated regions of stem cell-specific promoters in human iPS cells derived from extra-embryonic amnions and lung fibroblasts.

BACKGROUND: Human induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived. Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of "stemness" remain unknown in iPS cells derived from extra-embryonic and embryonic cells. METHODOLOGY/PRINCIPAL FINDINGS: We examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illumina's Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells. CONCLUSIONS/SIGNIFICANCE: We show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for "stemness" and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications.

Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products.

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.