Hajime Tsunoda

Improved explant method to isolate umbilical cord-derived mesenchymal stem cells and their immunosuppressive properties.

The umbilical cord (UC) has become one of the major sources of mesenchymal stem cells (MSCs). The common explant method of isolating UC-derived MSCs (UC-MSCs) involves mincing the UCs into small fragments, which are then attached to a culture dish bottom from which the MSCs migrate. However, the fragments frequently float up from the bottom of the dish, thereby reducing the cell recovery rate. To overcome this problem, we demonstrate an improved explant method for UC-MSC isolation, which involves the use of a stainless steel mesh (Cellamigo(®); Tsubakimoto Chain Co.), to protect the tissue from floating after the minced fragments are aligned at regular intervals in culture dishes. The culture medium was refreshed every 3 days and the adherent cells and tissue fragments were harvested using trypsin. The number of UC-MSCs isolated from 1u2009g of UC using the explant method with Cellamigo was 2.9 ± 1.4 × 10(6)/g, which was significantly higher than that obtained without Cellamigo (0.66 ± 0.53 × 10(6)/g) (n = 6, p < 0.01) when cells reached 80-90% confluence. In addition, the processing and incubation time required to reach 80-90% confluence was reduced in the improved explant method compared with the conventional method. The UC-MSCs isolated using the improved method were positive for CD105, CD73, CD90, and HLA class I expression and negative for CD45 and HLA class II expression. The isolated UC-MSCs efficiently inhibited the responder T cells induced by allogeneic dendritic cells in a mixed lymphocyte reaction. Conclusively, we demonstrated that the use of Cellamigo improves the explant method for isolating UC-MSCs.

Human Papillomavirus Genotype Distribution in Cervical Intraepithelial Neoplasia Grade 2/3 and Invasive Cervical Cancer in Japanese Women

OBJECTIVEnHuman papillomavirus vaccines are being introduced worldwide and are expected to reduce the incidence of cervical cancer. Here we report a cross-sectional study using a validated human papillomavirus genotyping method to reveal the human papillomavirus prevalence and genotype distribution in Japanese women with cervical intraepithelial neoplasia Grade 2/3 and invasive cervical cancer.nnnMETHODSnCervical exfoliated cells were collected from 647 patients with abnormal cervical histology (cervical intraepithelial neoplasia Grade 2, n = 164; cervical intraepithelial neoplasia Grade 3, n = 334; and invasive cervical cancer, n = 149), and subjected to the PGMY-PCR-based genotyping assay. The association between human papillomavirus infection and lesion severity was calculated using a prevalence ratio.nnnRESULTSnOverall, the prevalence of human papillomavirus deoxyribonucleic acid was 96.3% in cervical intraepithelial neoplasia Grade 2, 98.8% in cervical intraepithelial neoplasia Grade 3 and 88.0% in invasive cervical cancer (97.8% in squamous cell carcinoma and 71.4% in adenocarcinoma). The three most prevalent types were as follows: human papillomavirus 16 (29.3%), human papillomavirus 52 (27.4%) and human papillomavirus 58 (22.0%) in cervical intraepithelial neoplasia Grade 2; human papillomavirus 16 (44.9%), human papillomavirus 52 (26.0%) and human papillomavirus 58 (17.4%) in cervical intraepithelial neoplasia Grade 3; and human papillomavirus 16 (47.7%), human papillomavirus 18 (23.5%) and human papillomavirus 52 (8.7%) in invasive cervical cancer. The prevalence ratio of human papillomavirus 16 was significantly higher in cervical intraepithelial neoplasia Grade 3 compared with cervical intraepithelial neoplasia Grade 2 (prevalence ratio, 1.62; 95% confidence interval, 1.26-2.13) and in squamous cell carcinoma compared with cervical intraepithelial neoplasia Grade 3 (prevalence ratio, 1.55; 95% confidence interval, 1.25-1.87). Multiple infections decreased from cervical intraepithelial neoplasia Grade 2/3 (38.4/29.6%) to invasive cervical cancer (14.1%), whereas co-infections with human papillomavirus 16/52/58 were found in cervical intraepithelial neoplasia Grade 2/3.nnnCONCLUSIONSnThe results of this study provide pre-vaccination era baseline data on human papillomavirus type distribution in Japanese women and serve as a reliable basis for monitoring the future impact of human papillomavirus vaccination in Japan.

Neurosphere formation enhances the neurogenic differentiation potential and migratory ability of umbilical cord-mesenchymal stromal cells

BACKGROUND AIMSnThe human umbilical cord (UC) is a rich source of mesenchymal stromal cells (MSCs), which have been reported to have multi-lineage potential. The objectives of this study were to investigate the characteristics and capacity of UC-MSC neurosphere formation and whether this event enhances the propensity of UC-MSCs to undergo neural differentiation.nnnMETHODSnUC-MSCs were collected by the improved explant method. UC-MSCs and neurosphere-forming UC-MSCs (UC-MSC-neurospheres) were induced to undergo neurogenic differentiation, the latter of which were induced by suspension culturing in the presence of epidermal growth factor and basic fibroblast growth factor. The differentiation and migratory capacities of the individual cultures were then compared on the basis of the expression of neural markers, as measured by immunocytochemistry, immunoblotting and quantitative real-time polymerase chain reaction and transwell assays, respectively.nnnRESULTSnBoth UC-MSCs and UC-MSC-neurospheres were capable of differentiating into neurogenic cells when cultured in neurogenic differentiation medium. However, pre-conditioned UC-MSC-neurospheres exhibited significantly higher expression of neural markers--including microtubule-associated protein (MAP2), MUSASHI1, glial fibrillary acidic protein (GFAP), and NESTIN--compared with those derived from UC-MSCs directly. Moreover, UC-MSC-neurospheres expressed significantly higher levels of the stemness markers NANOG, KLF4 and OCT4 than did UC-MSCs. Migration assays also revealed that both UC-MSCs and UC-MSC-neurospheres actively migrate toward glucose-depleted cells.nnnCONCLUSIONSnNeurogenic differentiation potential probably is greater in UC-MSC-neurospheres than in UC-MSCs. Thus, UC-MSC-neurospheres may serve as a better source of cells for neurogenic regenerative medicine.

Serum- and xeno-free cryopreservation of human umbilical cord tissue as mesenchymal stromal cell source

BACKGROUND AIMSnHuman umbilical cord (UC) has become a notable source for mesenchymal stromal cells (MSCs) that can migrate to areas of inflammation and damaged tissue and can suppress excess immune reactions and to repair, respectively. Although UC is a solid tissue, there are several advantages, including repeatable uses from the same donor sample when needed and the possibility of future explorations for cells with unknown potential, if we could cryopreserve the UC as a living tissue material. However, because the cryoprotectants in the previous reports included animal- or allogeneic human-derived serum or no serum, the frozen-thawed UC-MSCs were inferior to fresh UC-MSCs in cell proliferation. The objective of this study was to find a suitable cryopreservation method of UC for clinical use.nnnMETHODSnThe UC was cut in cross-section and incised longitudinally, immersed in the cryoprotectant and frozen slowly. Later, it was thawed and minced rapidly, and the fragments of UC were cultured by improved explant method.nnnRESULTSnThe highest yield of cells was obtained from frozen-thawed UC with serum- and xeno-free cryoprotectant, STEM-CELLBANKER, when compared with others. The cells derived from frozen-thawed UC stored in STEM-CELLBANKER expressed the phenotypes of MSCs, retained the immunosuppressive properties in allogeneic mixed lymphocyte reactions and the differentiation potentials (into adipocyte and chondrocytes) comparable to those derived from fresh UC.nnnCONCLUSIONSnUC can be cryopreserved in serum- and xeno-free cryoprotectant as a living tissue while keeping its growth and functions equivalent to fresh UC. Our method is simple and feasible for clinical use.

Stage-specific embryonic antigen 4 in Wharton's jelly-derived mesenchymal stem cells is not a marker for proliferation and multipotency.

BACKGROUNDnUmbilical cord Whartons jelly (WJ) is a rich source of mesenchymal stem cells (MSCs) similar to bone marrow (BM) and adipose tissues. Stage-specific embryonic antigen (SSEA)4 has been reported as a stem cell marker in BM-derived MSCs, but whether SSEA4(+) cells have growth and differentiation advantages over SSEA4(-) cells remains controversial. To gain insight into the role of SSEA4, we studied SSEA4(+) cells in WJ-derived MSCs (WJ-MSCs).nnnMETHODSnWJ-MSCs were collected by the explant (WJe-MSCs) or collagenase methods (WJc-MSCs) and analyzed by flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR). To evaluate whether culture conditions influenced the SSEA4 expression, WJe-MSCs were cultured in the medium supplemented with different fetal bovine serum (FBS) concentrations.nnnRESULTSnSSEA4 was expressed for a long-term culture. In contrast, SSEA3(+) disappeared rapidly in early passages of the culture. The incidence of SSEA4(+) and SSEA3(+) cells was similar between WJe-MSCs and WJc-MSCs at passages P0-P9, except for transient depletion of SSEA4 expression in early passages of WJe-MSCs. These were CD73(+)CD105(+) cells that express embryonic stem cell markers detected by RT-PCR. No differences in growth and differentiation ability of osteocytes and adipocytes were observed between the sorted SSEA4(+) cells and SSEA4(-) cells. Further, SSEA4 expression in WJe-MSCs was significantly correlated with FBS concentration in the culture medium.nnnDISCUSSIONnSSEA4, which may display altered expression profiles in response to culture conditions, may not be an essential marker of WJ-MSC multipotency.

Genotype Distribution of Human Papillomaviruses in Japanese Women with Abnormal Cervical Cytology

We report the prevalence and genotype distribution of human papillomaviruses (HPVs) among Japanese women with abnormal cervical cytology using the PGMY-CHUV assay, one of PGMY-PCR-based lineblot assays that was validated and shown to be suitable for the detection of multiple HPV types in a specimen with minimum bias. Total DNA was extracted from cervical exfoliated cells collected from 326 outpatients with abnormal Pap smears. Overall, 307 specimens (94%) were HPV-positive, 30% of which contained multiple genotypes. The prevalence of HPV DNA was 83% (49/59 samples) in atypical squamous cells of undetermined significance (ASC-US); 91% (20/22 samples) in atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); 97% (130/134 samples) in low-grade squamous intraepithelial lesion (LSIL); and 99% (85/86 samples) in high-grade squamous intraepithelial lesion (HSIL). Three most frequent HPV types detected in HSIL were HPV16 (36%), HPV52 (24%), and HPV58 (14%). Our results suggest that multiple HPV infections are more prevalent in Japanese women than previously reported, and confirm that HPV52 and 58 are more dominant in their cervical precancerous lesions when compared to those reported in Western countries.

Immunosuppressive properties of Wharton's jelly-derived mesenchymal stromal cells in vitro.

AbstractRecent studies have reported nthat mesenchymal stromal cells (MSCs) migrate to areas of inflammation and suppress adverse immune reactions. Bone marrow (BM)-derived MSCs have been successfully used in patients with acute graft versus host disease (GVHD), but the harvesting of BM carries certain risks for the donor. To circumvent these, we obtained MSCs from Wharton’s jelly (WJ) derived from umbilical cord and investigated their potential for immunosuppression. In a mixed lymphocyte reaction (MLR), responder T cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by WJ-MSCs derived from the same donor of responder cells or those from a third party donor. These inhibitory effects were reversed in a dose-dependent manner in the presence of 1-methyl-DL-tryptophan, an inhibitor of the soluble factor indoleamine 2, 3-dioxygenase (IDO). Immunosuppression by WJ-MSCs was also attenuated by blocking cell–cell contact between WJ-MSCs and responder T cells using a Transwell chamber. Moreover, IDO gene expression was induced in both WJ- and BM-MSCs by inflammatory cytokine IFN-γ, but HLA-DR was expressed in BM-MSCs and not in WJ-MSCs upon stimulation by a relatively low concentration of IFN-γ. These results indicate that WJ-MSCs exert their immunosuppressive effects by cell–cell contact with activated T cells and in part through IDO, and suggest the need for cells rather than soluble factors secreted from MSCs to achieve immunosuppressive therapy in severe cases of GVHD.

Intravenous injection of umbilical cord-derived mesenchymal stromal cells attenuates reactive gliosis and hypomyelination in a neonatal intraventricular hemorrhage model

Intraventricular hemorrhage (IVH) is a frequent complication of preterm newborns, resulting in cerebral palsy and cognitive handicap as well as hypoxic ischemic encephalopathy and periventricular leukomalacia. In this study, we investigated the restorative effect on neonatal IVH by umbilical cord-derived mesenchymal stromal cells (UC-MSCs) cultured in serum-free medium (RM medium) for clinical application. UC-MSCs were cultured with αMEM medium supplemented with FBS or RM. A neonatal IVH mouse model at postnatal day 5 was generated by intraventricular injection of autologous blood, and mice were intravenously administered 1×105 UC-MSCs two days after IVH. Brain magnetic resonance imaging was performed at postnatal day 15, 22 and neurological behavioral measurements were performed at postnatal day 23, accompanied by histopathological analysis and cytokine bead assays in serum after IVH with or without UC-MSCs. Both UC-MSCs cultured with αMEM and RM met the criteria of MSCs and improved behavioral outcome of IVH mice. Moreover the RM group exhibited significant behavioral improvement compared to the control group. Histopathological analysis revealed UC-MSCs cultured with RM significantly attenuated periventricular reactive gliosis, hypomyelination, and periventricular cell death observed after IVH. Furthermore, human brain-derived neurotrophic factor and hepatocyte growth factor were elevated in the serum, cerebrospinal fluid and brain tissue of neonatal IVH model mice 24h after UC-MSCs administration. These results suggest UC-MSCs attenuate neonatal IVH by protecting gliosis and apoptosis of the injured brain, and intravenous injection of UC-MSCs cultured in RM may be feasible for neonatal IVH in clinic.

Two cases of adenocarcinoma in situ arising in lobular endocervical glandular hyperplasia indicating localization of mucin on the cluster surface as an early cytological finding of malignant transformation: OKUYAMA et al.

Lobular endocervical glandular hyperplasia (LEGH) is an endocervical glandular hyperplastic lesion containing pyloric gland‐like mucin, and has recently been recognized as a precursor lesion of malignant glandular lesions of the endocervix. The pyloric gland‐like mucin contained in LEGH and gastric‐type adenocarcinoma is observed as golden‐yellowish by Papanicolaou staining. However, to our knowledge, the chronological course of the endocervical cytology of LEGH, eventually resulting in malignancy, has never been demonstrated to date. Here, we report two cases of gastric‐type adenocarcinoma in situ (AIS) arising in LEGH, together with an analysis of their cytological course. In both cases, localization of mucin on the surface of glandular cell clusters was observed prior to nuclear atypia in endocervical cytology. In addition, the diagnosis of gastric‐type AIS arising in LEGH was confirmed by pathological diagnosis of hysterectomy specimens in both cases. Histologically, all glandular cells of the LEGH without nuclear atypia contained a large amount of PAS‐positive mucin. On the other hand, in atypical glandular cells, localization of the mucin on the luminal surface was observed, although mucin was abundant throughout the cytoplasm in some areas. Our cases show the course of acquirement of cytological atypia of LEGH, and indicate the significance of localization of mucin on the surface of glandular cell clusters as an early finding of the malignant transformation of LEGH in endocervical cytology. Our results indicate that the distribution of mucin in glandular cells should be analyzed together with nuclear atypia in the endocervical cytology of suspected cases of LEGH.

Uterine adenomatoid tumor containing epithelioid oxyphilic cells showing prominent down-expression of HBME1 and increased Ki-67 labeling index: Letter to the Editor

To the Editor: Adenomatoid tumors (ATs) are benign tumors, generally composed of variable-sized tubules formed by flat cells of mesothelial origin, along with muscular hypertrophy. Uterine ATs composed of epithelioid oxyphilic cells of mesothelial origin are rare. A 31-year-old woman (gravida 0) visited the gynecology department of our hospital for the first time because of dysmenorrhea. She had no obvious history of asbestos exposure, and was not an immunosuppressed patient. Magnetic resonance imaging displayed a 3 cm uterine intramuscular mass, which was suspected as being uterine leiomyoma. Although she was once treated with low-dose estrogen-progestin (LEP), LEP treatment was discontinued because deep vein thrombosis (DVT) was clinically highly suspected. Then, she was followed-up with a prescription of non-steroidal anti-inflammatory drugs (NSAIDs); however, her abdominal pain continued. Thus, her uterine mass was enucleated 3 years and 3 months after her first visit, and subsequently, the mass was pathologically examined. Histologically, cuboid cells with round small nuclei and oxyphilic cytoplasm were presented in a funicular or solid pattern along with a few flat cells forming gland-like vacuoles with thin bridging strands at the periphery of the mass (Fig. 1a,b). Mitosis of the epithelioid oxyphilic cells was not observed in any of 50 high-power fields. At the center of the mass, proliferating flat cells forming glandlike vacuoles were found among the proliferated smooth muscle cells, and the ratio of epithelioid oxyphilic cells to the flat cells gradually decreased upon approaching the center. Lymphoid aggregates were never observed in the mass. Immunohistochemical analyses demonstrated that flat cells forming gland-like vacuoles among the smooth muscle cells were positive for D2-40 (Fig. 1c), WT-1, calretinin, cytokeratin AE1/AE3, and BRCA1-associated protein 1 (BAP1), and were negative for CD34, epithelial membrane antigen (EMA), and desmin; these results indicated that this tumor was AT. Furthermore, epithelioid oxyphilic cells were immunohistochemically positive for D2-40 (Fig. 1c), WT-1, calretinin, cytokeratin AE1/AE3, and BAP1, and were negative for PAX8, CD34, EMA, desmin, BerEP4, CEA, CD56, chromogranin A, synaptophysin, inhibin, glucose transporter-1 (GLUT-1), CD146, and insulin-like growth factor II mRNA-binding protein 3 (IMP3). Remarkably, HBME1 was expressed in the flat cells, but was weakly expressed in only a few of the epithelioid oxyphilic cells; almost all were negative (Fig. 1d). The Ki-67 labeling index was about 6.7% (35/ 521) in the epithelioid oxyphilic component, which was higher than that of flat cells, which was about 1.3% (7/ 531) (Fig. 1e). Malignant mesothelioma was denied because of positivity for BAP1 and negativity for GLUT-1, CD146, and IMP3. We hence diagnosed this uterine tumor as AT containing epithelioid oxyphilic cells. After the operation, the tenderness detected upon gynecological examination and peritoneal thickening of the Douglas pouch were improved. She is alive without recurrence at 4 years and 10 months after her first visit. Epithelioid oxyphilic AT in the uterus, which is the most common site for AT, are rare; a study of AT including a case of uterine AT with epithelioid oxyphylic cells was published more than 30 years earlier. However, the immunohistochemical characteristics were not analyzed at all in that report. Because AT shows site-specific morphological and immunohistochemical diversity, immunohistochemical analyses of uterine epithelioid oxyphilic AT is of high importance. The epithelioid oxyphilic cells were of mesothelial origin because of immunohistochemical positivity for mesothelial markers, such as WT-1, D2-40, and calretinin. Because many flat cells, which are usually observed in AT, were also found in the center of the uterine mass, this tumor was suspected to be AT with epithelioid oxyphilic cells; however, malignant mesothelioma remained to be a differential diagnosis. It has been reported that the loss of BAP1 expression is a high sensitive and specific finding of malignant mesothelioma including peritoneal malignant mesothelioma. In our case, epithelioid oxyphilic cells were positive for BAP1, which indicated oxyphilic cells as benign mesothelial-origin cells rather than malignant mesothelioma. Thus, we diagnosed this uterine tumor as AT containing epithelioid oxyphilic cells. In our case, because the Ki-67 labeling index was higher in epithelioid oxyphilic cells than in flat cells, epithelioid oxyphilic cells appeared to have increased proliferation potential. Furthermore, HBME1 labeled flat cells of the AT in our case, similar to HBME1 expression in usual ATs; however, epithelioid oxyphilic cells in the same tumor showed remarkably decreased expression of HBME1 as a relatively highly sensitive mesothelial cell marker. These immunohistochemical findings suggested the high proliferative potential of epithelioid oxyphilic cells of our AT case, and this might be owing to a slightly decreased rate of