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

BACKGROUND AIMS: Monocytes, 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. METHODS: To 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. RESULTS: The 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. CONCLUSIONS: The 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.

Improved Production of Induced Pluripotent Stem Cells Using Dot Pattern Culture Plates.

The rate of cell proliferation is a crucial factor in cell production under good manufacturing practice (GMP) control. In this study, we identified a culture system for induced pluripotent cells (iPSCs) that supports cell proliferation and viability and maintains the cells in an undifferentiated state even at 8 days after seeding. This system involves the use of dot pattern culture plates that have been coated with a chemically defined scaffold which has high biocompatibility. Under cell starvation conditions, where medium exchange was not performed for 7 days or where the amount of medium exchange was reduced to half or a quarter, iPSC viability and lack of differentiation were maintained. The rate of cell viability in this culture system was greater than generally obtained by standard culture methods. The cells in this compartmentalized culture system could be induced to differentiate in a controlled and consistent manner: differentiation of endoderm occurred in a controlled and consistent manner: endoderm, mesoderm, and ectoderm could be consistently induced to differentiate in the cultures. In conclusion, we have developed a culture system that supports high viability in iPSCs and allows their controlled differentiation. This system has the potential for use in GMP-based production of iPSCs for clinical purposes.

Adhesion Characteristics of Human Pancreatic Islets, Duct Epithelial Cells, and Acinar Cells to a Polymer Scaffold.

We reported in 2018 that among several extracellular matrices, fibronectin, type I collagen, type IV collagen, laminin I, fibrinogen, and bovine serum albumin, fibronectin is particularly useful for adhesion of porcine pancreatic tissue. Subsequently, we developed a technology that enables the chemical coating of the constituent motifs of fibronectin onto cell culture dishes. In this experiment, we used islets (purity ≥ 90%), duct epithelial cells (purity ≥ 60%), and acinar cells (purity ≥ 99%) isolated from human pancreas according to the Edmonton protocol published in 2000 and achieved adhesion to the constituent motifs of fibronectin. A solution including cGMP Prodo Islet Media was used as the assay solution. In islets, adhesion was enhanced with the constitutive motifs of fibronectin compared with uncoated islets. In the functional evaluation of islets, insulin mRNA expression and insulin secretion were enhanced by the constitutive motif of fibronectin compared with non-coated islets. The stimulation index was comparable between non-coated islets and fibronectin motifs. In duct epithelial cells, adhesion was mildly promoted by the fibronectin component compared with non-coated component, while in acinar cells, adhesion was inhibited by the fibronectin component compared with the non-coated component. These data suggest that the constitutive motifs of fibronectin are useful for the adhesion of islets and duct epithelial cells.

A chemically-defined plastic scaffold for the xeno-free production of human pluripotent stem cells.

Clinical use of human pluripotent stem cells (hPSCs) is hampered by the technical limitations of their expansion. Here, we developed a chemically synthetic culture substrate for human pluripotent stem cell attachment and maintenance. The substrate comprises a hydrophobic polyvinyl butyral-based polymer (PVB) and a short peptide that enables easy and uniform coating of various types of cell culture ware. The coated ware exhibited thermotolerance, underwater stability and could be stored at room temperature. The substrate supported hPSC expansion in combination with most commercial culture media with an efficiency similar to that of commercial substrates. It supported not only the long-term expansion of examined iPS and ES cell lines with normal karyotypes during their undifferentiated state but also directed differentiation of three germ layers. This substrate resolves major concerns associated with currently used recombinant protein substrates and could be applied in large-scale automated manufacturing; it is suitable for affordable and stable production of clinical-grade hPSCs and hPSC-derived products.

Levels of Physical Fitness and Weight Status in Children and Adolescents: A Comparison between China and Japan.

This study compared the physical fitness level and weight status of children and adolescents in China and Japan. Children and adolescents aged 7-18 years were recruited (China: n = 5660; Japan: n = 5660). Physical fitness was assessed using seven core items-grip strength, 30-s sit-ups, standing long jump, sit-and-reach, 20-s repeated straddling, 20-m shuttle run test, and 50-m dash. A physical fitness index (PFI) was calculated by adding all items' Z-scores. We conducted comparisons of PFI and its distribution, each physical fitness item, and weight status for individuals from China and Japan across all ages. The PFI was lower in China than in Japan for all age groups, with an especially large difference at age 18 years for boys (a difference of 9.05) and girls (a difference of 9.10) (p < 0.001). The same result was seen for the seven items. The PFI distribution for children and adolescents was more disperse among those in Japan than among those in China. Obesity prevalence was 2.84 times higher in China than in Japan. An inverted U-shaped relationship was observed between physical fitness and nutritional status. Children and adolescents showed markedly lower physical fitness and higher obesity prevalence in China than in Japan.

Comparison of growth and nutritional status of Chinese and Japanese children and adolescents.

BACKGROUND: The difference in growth and nutritional status, both important indices of population quality, between Chinese and Japanese children and adolescents is unknown. AIM: This study aimed to compare growth and nutritional status between Chinese and Japanese children and adolescents. SUBJECTS AND METHODS: The height-for-age and BMI-for-age distribution of 9,226 children and adolescents aged 7-18 years from China and Japan were described with the Lambda Mu and Sigma method. Wasting, overweight and obesity were evaluated based on BMI-for-age cut-offs of the 2007 WHO Child Growth Reference. RESULTS: For boys, the overall average height, weight and BMI of Chinese participants were 3.0 cm, 4.8 kg and 1.2 kg/m2 greater compared with Japanese participants, respectively; for girls, these were 4.6 cm, 3.9 kg and 0.6 kg/m2, respectively. Compared with Japanese children, the 3rd, 50th and 97th percentiles of height-for-age, 1Z-score, and 2Z-score of BMI-for-age of Chinese children were greater, whereas the minus 2Z-scores of Chinese children were less. The prevalence of wasting, overweight and obesity among Chinese participants was greater. CONCLUSIONS: Compared with Japanese children, Chinese children tended to be taller. The worrying burden of overweight, obesity and wasting was recognised among Chinese children.