Kaoru Kita

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell–like properties†‡

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti‐apoptotic properties compared with those of non‐SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 × 10 3 SP cells were sufficient for tumor formation, whereas an injection of 1 × 10 6 non‐SP cells did not initiate tumors. Re‐analysis of SP cell–derived tumors showed that SP cells generated both SP and non‐SP cells and tumor‐initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non‐SP cells, and several so‐called “stemness genes” were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy. (HEPATOLOGY 2006;44:240–251.)

Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules.

Abstract Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.

Testosterone administration promotes regeneration of chemically impaired spermatogenesis in rats

Aim: It has been proposed that gonadotropin‐releasing hormone (GnRH) analog administered after testicular damage stimulates the recovery of spermatogenesis. However, GnRH analogs suppress the function of sex accessory organs. In this study, we investigated whether testosterone also stimulates the regeneration of rat spermatogenesis after exposure to busulfan.

Ectopic porcine spermatogenesis in murine subcutis: tissue grafting versus cell-injection methods.

Fragments of testis tissue from immature animals grow and develop spermatogenesis when grafted onto subcutaneous areas of immunodeficient mice. The same results are obtained when dissociated cells from immature testes of rodents are injected into the subcutis of nude mice. Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient (SCID) and NOD/Shi-SCID, IL-2Rgammacnull (NOG) mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell-injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not significant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals.

Primary Synovial Sarcoma of the Kidney

The case was a 40-year-old female. She visited a local doctor with a chief complaint of right side abdominal pain. A right kidney tumor measuring 10 cm in diameter was observed in an abdominal Computed Tomography (CT) scan. Based on the CT image, the possibility of angiomiolipoma (AML) could not be ruled out, but a high maximum standardized uptake value (SUVmax) of 7.8 was observed in a Positron Emission Tomography CT (PET-CT) scan and there was a possibility of malignancy. We therefore performed a transperitoneal right radial nephrectomy. Although adhesion of the tumor to the duodenum and the inferior vena cava was observed, it was possible to perform an excision. The tumor accounted for a large proportion of the excised kidney; the surrounding areas had taken on a cyst-like structure, and the interior comprised grayish brittle tissue exhibiting solid growth. Histologically, gland-like and cyst-like structures composed of cylindrical cuboidal cells and mainly characterized by the solid growth of short fusiform-shaped and oval-shaped basophilic cells were observed, and we believed it was a synovial sarcoma. There were no malignant findings in the adrenal gland. There have been approximately 30 reported cases around the world of synovial sarcoma that developed in the kidney, and we herein report this case with bibliographic considerations.

Neuroendocrine Carcinoma of the Bladder.

The case was a 67-year-old male who visited our hospital with a major complaint of macroscopic hematuria. A bladder tumor was found. When a transurethral resection of the bladder tumor was performed, the histopathological diagnosis was neuroendocrine bladder cancer. After chemotherapy with cisplatin and etoposide a partial shrinkage of the tumor was observed; however, the patient expired 7 months after the first visit.

Proliferation of spermatogonial stem cells and spermatogenesis in vitro

Detection of spermatogonial stem cells (SSC) became possible 10 years ago, with the transplantation of germ cells into the seminiferous tubules of mice. Using this assay system, attempts to maintain and expand SSC in vitro finally bore fruit. Gonocytes from neonatal mice and spermatogonial stem cells from adult mice were plated on feeder cells in a medium supplemented with Glial cell line-derived neurotrophic factor (GDNF) along with certain other factors. The germ cells that emerged under such conditions, named germline stem (GS) cells, proliferated exponentially through self-renewing division. GS cells in vitro show features of differentiation as well. Some expressed c-kit, which is a cell surface marker of differentiating spermatogonia. Therefore, it seems that GS cells undergo both self-renewing and differentiating cell divisions in vitro. There is a century of history behind attempts to reproduce spermatogenesis in vitro and significant progress has been made. Nonetheless, there are few established culture-based protocols for recreating spermatogenesis in vitro. GS cells would be an ideal starting material in this regard.