Masaharu Mori

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.

Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells.

Recombination activating genes (RAG-1 and RAG-2), involved in V(D)J rearrangement of immunoglobulin genes, have been thought to be expressed only in immature stages of B-cell development. However, RAG-1 and RAG-2 transcripts were found to be reexpressed in mature mouse B cells after culture with interleukin-4 in association with several different co-stimuli. Reexpression was also detected in draining lymph nodes from immunized mice. RAG-1 and RAG-2 proteins could be detected by immunofluorescence microscopy in the nuclei of B cells cultured in vitro and in the germinal centers of draining lymph nodes. These findings suggest that RAG gene products play a heretofore unsuspected role in mature B cells.

Role of the macrophage in erythropoiesis

Macrophages, which are derived from precursor cells in the bone marrow, differentiate specifically under the influence of the local microenvironment. Resident macrophages in hematopoietic tissues can be distinguished from other stromal cells and monocytes by immunostaining with monoclonal antibody F4/80 and anti‐Forssman glycosphingolipid antibody, respectively. Erythroid colony‐forming units adhere to a resident macrophage and differentiate to erythroblasts in the presence of erythropoietin (EPO), resulting in the formation of an erythroblastic island. Resident macrophages play a supportive role in erythropoiesis, probably by preventing apoptosis of the erythroid precursors via adhesive interaction between very late activation antigen 4 and vascular cell adhesion molecule 1. Herein is proposed a model of erythropoiesis based on cooperative interaction between EPO and resident macrophages.

Two human myeloma cell lines, amylase-producing KMS-12-PE and amylase-non-producing KMS-12-BM, were established from a patient, having the same chromosome marker, t(11;14)(q13;q32)

Two human myeloma cell lines, KMS‐12‐PE and KMS‐12‐BM, were established from a 64‐year‐old woman with a non‐producing type of multiple myeloma. The KMS‐12‐PE line originated from the pleural effusion and the KMS‐12‐BM from the bone marrow. These two lines showed the same chromosome marker, t(11;14)(q13;q32). However, their phenotypes of surface markers differed from each other. KMS‐12‐BM cells were positive to CD20, CD38 and PCA‐1, showing the plasmacytoid (immature plasma cell) stage of B‐cell differentiation, while KMS‐12‐PE cells were positive to CD38 and PCA‐1, but not to CD20, indicating the terminal differentiated stage of B‐cells. As seen in the pleural effusion of the patient, KMS‐12‐PE cells ectopically produced a salivary type of amylase, but KMS‐12‐BM cells did not. Interestingly, the chromosome abnormality of del(1)(p22→pter) near the region of Ip21, where the amylase gene was assigned, was noticed in as many as 76% of KMS‐12‐PE cells.

Establishment of five human myeloma cell lines

SummaryFive human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.

Epithelioid trophoblastic tumor: morphological and immunohistochemical study of three lung lesions.

Epithelioid trophoblastic tumor (ETT) is a term proposed for an unusual variant of trophoblastic tumor that is closely related to choriocarcinoma but shows monomorphic growth of highly atypical trophoblastic cells instead of the typical dimorphic pattern of choriocarcinoma. We report here 3 cases of ETT, all of which were lung lesions probably originating from uterine trophoblastic disease. The antecedent pregnancies of the 3 cases were hydatidiform mole, invasive mole, and term pregnancy, respectively. The tumors were composed of highly atypical mononucleate cells, which mainly involved alveolar spaces, forming nests with central eosinophilic necrosis. Multinucleate giant cells were found within the nests, but they were fewer in number than in typical choriocarcinoma. The tumors were not associated with extensive hemorrhage or necrosis, except for 1 case, in which the ETT was combined with typical dimorphic choriocarcinoma. Immunohistochemically, multinucleate giant cells and occasional mononucleate tumor cells showed positivity for human chorionic gonadotropin. Staining for human placental lactogen was positive in rare multinucleate giant cells, and in 1 case, tumor cells showed diffuse positivity for placental alkaline phosphatase. Because ETT has a remarkably epithelioid appearance in cytological and architectural features, differentiation from the epithelial malignancies is problematic. Trophoblastic markers are frequently expressed in nontrophoblastic tumors, and reactivity for those markers alone is not sufficient for exclusion of other tumors. Rather, evidence of ETT comes from a combination of morphological features, immunohistochemical study, and clinical history.

1,4-Naphthoquinone is a potent inhibitor of human cancer cell growth and angiogenesis

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. Vitamin K2 and K3, which are naphthoquinone derivatives, inhibit angiogenesis. We examined the anti-cancer and anti-angiogenic effects of naphthoquinones and its structurally related compounds. Among these 13 compounds, 1,4-naphthoquinone strongly inhibited both human colon cancer cell (HCT116) growth and angiogenesis. To clarify the anti-angiogenic mechanism, the effects of 1,4-naphthoquinone on human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and chemotaxis were examined. Consequently, 1,4-naphthoquinone inhibited HUVEC functions. These results suggest that 1,4-naphthoquinone may be useful to cancer therapy.

CAPILLARY GROWTH FROM REVERSED RAT AORTIC SEGMENTS CULTURED IN COLLAGEN GEL

The process of angiogenesis from aortic segments turned inside out and embedded in collagen gel was studied. Two to three days after inoculation, fibroblastic cells migrated from both ends of the segments. Later, capillary sprouts also appeared from both ends of the segments but not from the outer surface, even though there was a covering of endothelial cells. If the outer surface was injured, capillaries sometimes appeared at the damaged site. This may suggest that endothelial cells have more affinity for basement membrane than collagen gel and that they migrate only from an injured site. Immuno‐histochemical staining demonstrated factor VHI‐related antigen in the capillary structures but not in the fibroblastic cells. Electron microscopically, capillary lumina were lined with several endothelial cells, and fibroblastic cells had the characteristics of smooth muscle cells. Since these fibroblastic cells have been known to appear under angiogenetic conditions in vivo, they may play an important role in angiogenesis, and the present culture technique may be a useful model for studying this process. ACTA PATHOL JPN 38: 1503∼1512, 1988.

Capillary growth of rat aortic segments cultured in collagen gel without serum.

The process of angiogenesis was studied under serum‐free conditions using rat aortic segments in three‐dimensional collagen gel. In serum‐free and growth factor free conditions, the capillaries formed networks and tube‐like structures, and the endothelial cells produced von Willebrand factor, laminin and type IV collagen, but the number of capillaries was lower and their growth was slower than in medium containing 20% fetal calf serum (FCS). Incorporation of bromodeoxyuridine (BrdU) and inhibition of growth by hydroxyurea suggested that capillary growth depended mainly on cell proliferation and not on migration. Capillary growth in PRMI 1640 or DM EM was similar and more efficient than in MEM. Only slight growth was seen in Medium 199 and HAM‐F12. The addition of serum to the medium accelerated capillary growth in proportion to the amount added. In serum‐free conditions, ITS(+) and fibroblast growth factor (FGF) promoted capillary growth, but not to a significant extent. There ware no differences in capillary growth among the gel matrices used (type I collagen, type I+ 11 collagen, type I IV collagen, fibrin and plasma clot).

Establishment of Lymphotoxin β Receptor Signaling-Dependent Cell Lines with Follicular Dendritic Cell Phenotypes from Mouse Lymph Nodes

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin β receptor mAb and TNF-α. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, FcγRIIB, lymphotoxin β receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.