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.

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.

Inflammatory pseudotumor of the kidney with renal artery penetration.

Inflammatory pseudotumor (IPT) is a quasineoplastic lesion that most commonly involves the lung and the orbit; kidney involvement is rare. We report a case of inflammatory pseudotumor of the kidney. The patient was a 61-year-old man who presented with no symptoms. Nonenhanced computed tomography (CT) demonstrated an ill-defined, isodensity mass measuring 3.5 cm in the lower portion of the left kidney. Contrast-enhanced CT showed that branches of the renal artery without encasement penetrated the tumor; there was a little enhancement in the mass on the arterial phase and homogeneous enhancement on the venous phase. On magnetic resonance imaging the mass showed intermediate signal intensity on T1-weighted images (T1WIs) and low signal intensity on T2WIs. Most IPTs of the kidney appear as an ill-defined, hypovascular, homogeneous tumor on CT images, with variable signal intensity on MRI T1WIs and low signal intensity on T2WIs. Our case had the same imaging findings, with branches of the renal artery penetrating the tumor. If the renal tumor has these radiological findings, the tumor may be IPT.

Laparoscopic removal of Müllerian structures and orchiopexy for persistent Müllerian duct syndrome.

A 1-year-old boy presented for evaluation of bilateral undescended testes. Diagnostic laparoscopy was conducted, and uterine tissue with bilateral gonads was detected in the rectovesical fossa. Chromosomal analysis revealed a 46,XY karyotype. Persistent müllerian duct syndrome was diagnosed, and laparoscopic surgery was performed to treat this condition. The uterus was incised at a distal site and withdrawn through the port. The bilateral testes were fixed in the scrotum. The patient was discharged 3 days later. We believe that the laparoscopic approach is a valid, alternative choice to traditional surgery for resolution of this condition, permitting minimally invasive surgery.

Laparoscopic radical prostatectomy: initial cases at Okayama University Hospital.

We performed laparoscopic prostatectomy in seven cases with organ-confined prostate cancer. In 6 cases, the surgery was completed successfully and the mean operative time was 424 min. Volume of blood loss was 200 to 3,200 ml and catheterization lasted 6 to 37 days. No major complications were observed in 6 of the cases. In one case, open surgical conversion was necessary mainly due to a bladder injury. Although these were the first cases of laparoscopic prostatectomy in our institution, the technical difficulty and complexity of the surgery were moderate. We believe that laparoscopic radical prostatectomy will become a standard option for the treatment of organ-confined prostate cancer.