Kanji Miyamoto

Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells.

Various undifferentiated embryonic stem (ES) cells can grow on mouse embryonic fibroblast (MEF) feeders. However, the risk of zoonosis from animal feeders to human ES cells generally excludes the clinical use of these human ES cells. We have found that human placenta is a useful source of feeder cells for the undifferentiated growth of primate ES cells. As on MEF feeders, primate ES cells cultured on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) cells had undifferentiated growth. The cultured primate ES cells expressed Oct‐4, alkaline phosphatase, and SSEA‐4. The primate ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into severe combined immunodeficient mice. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications.

Transformation of CD8 + T‐Cells Producing a Strong Cytopathic Effect on CD4+ T‐Cells through Syncytium Formation by HTLV‐II

Human T‐cell leukemia virus type II (HTLV‐II) is thought to play an important role in the development of CD8 + T‐cell malignancies resembling hairy cell leukemia. In this study, dramatic cytopathic effects characterized by syncytium formation in various CD4+ T‐cell lines were observed upon their cocultivation with HTLV‐II infected T‐cells. The HTLV‐II infected T‐cells, however, did not die as a result of syncytium formation. HTLV‐II also transformed CD4 + T‐cells and CD8 + T‐cells at various coculture ratios. Furthermore, sera from antiHTLV‐II antibody‐positive specific carriers inhibited syncytium formation in the CD4+ T‐cells. These results suggest that HTLV‐II infection may contribute to the pathogenesis of associated CD8 + T‐cell malignancies.

A human T cell line with an abnormal trisomy 2 karyotype established by coculture of peripheral lymphocytes with an HTLV-II-infected simian leukocyte cell line

A new human T cell in with a chromosomal abnormality (47, XY, +2), designated AS‐IIA, was established by coculturing peripheral blood leukocytes of a healthy adult male with a lethally irradiated human T lymphotropic virus type II (HTLV‐II)‐infected simian leukocyted cell line (Si‐IIA). A polymerase chain reaction method showed that this interleukin‐2 (IL‐2)‐dependent cell line possessed the HTLV‐II provirus genome; the cells also reacted with HTLV‐II‐positive human sera, anti‐HTLV‐I/II p24, and anti‐HTLV‐II gp46 antibodies. AS‐IIA cells expressed the suppressor/cytotoxic T cell markers CD3+, CD4‐, CD25+, and HLA‐DR+, with later conversion ot CD8‐. These cells showed better proliferation than other human HTLV‐II‐infected cell lines with normal karyotypes, but were not transplantable into severe combined immunodeficiency mice. Virus production from AS‐IIA was confirmed not only by electron microscopic examination, which revealed mature and immature type C virus particels, but also by the capacity of the line to immortalize human T cells. These results suggest that HTLV‐II shows broad tropism for T cells including CD4+ or CD8+, and that not only SI‐IIA, but also AS‐IIA, are goode sources of HTLV‐II. The authors of the present study believe that AS‐IIA may be a useful human T cell line for the investigation of HTLV‐II in comarison with HTLV‐I.

Co-expression of CD4 and CD8 associated with elevated interleukin-4 in a cord T cell line derived by cocultivating normal cord leukocytes and an HTLV-II-producing simian leukocyte cell line (Si-IIA)

SummaryA new interleukin-2(IL-2)-dependent T cell line, designated CS-IIA, was established by cocultivating normal human cord leukocytes and a lethally X-irradiated HTLV-II-producing simian leukocyte cell line (Si-IIA). CS-IIA showed CD4 dominance during the early culture. However, after addition of IL-2, CS-IIA predominantly co-expressed CD4 and CD8 (69.5%) and also expressed the surface markers CD1−, CD3+, CD19−, CD25+ and HLA-DR+. A significantly elevated level of IL-4 (1697 pg/ml) was observed in the culture supernatant from CS-IIA. In addition, the conversion of phenotype from some CD4+CD8+ cells to CD4+CD8− was demonstrated by the neutralization assay using anti-IL-4 antibody. CS-IIA had a normal human karyotype and was free from Epstein-Barr virus nuclear antigen and immunoreactive with sera of HTLV-I- or HTLV-II-infected patients and anti-HTLV-1, p19 or p24 mAb. The provirus genome of HTLV-II was detected in this cell line by the polymerase chain reaction combined with a digoxigenin-enzyme-linked immunosorbent assay. However, electron microscopy of CS-IIA cells revealed no C-type virus particles in the extracellular space. These results indicate that HTLV-II can be transmitted from an HTLV-II-infected simian leukocyte cell line to human cord T lymphocytes and suggest that co-expression of CD4 and CD8 on T cells may be induced by the high level of IL-4, which can mediate CD8 induction on CD4+ T cell clones.

Chromosome analysis after ex vivo expansion of CD34+ cells from human cord blood

Ex vivo expansion of cord blood hematopoietic progenitors is an attractive way to prepare a sufficient number of transplantable cells for cord blood transplantation in adult patients. The expanded cells need to have genetic stability. Karyotypic analysis of the expanded cells from cord blood CD34(+) cells by 7-day culture with stem cell growth factor, interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor, and erythropoietin was performed. Several-fold increases in total cell number and CFU-GM were 186.7 +/- 62.1 and 27.1 +/- 9.4 (mean +/- standard deviation of data from 6 samples), respectively. Karyotypes were analyzed in 600 expanded cells in total, and all of them showed normal karyotypes. This observation suggests that multifactor supported expansion of cord blood cells may not induce karyotypic abnormality, although a limited number of ex vivo expanded cells were tested.

A B-CELL LINE HAVING CHROMOSOME 14 ABERRATION AT BREAK BAND qll DERIVED FROM AN ADULT T-CELL LEUKEMIA PATIENT

A B‐cell line having translocations of chromosome 14 at break band q11 (the assigned locus of the α‐chain gene of the T‐cell antigen receptor) and chromosome 3 at break band p25 (the assigned locus of the c‐raf‐1 oncogene) was established from peripheral blood leukocytes of an adult T‐cell leukemia (ATL) patient. The same chromosome 14 aberration at break band q11 and chromosome 3 aberration at break band p25 were also found in fresh T‐cell leukemia cells. The B‐cell line is surface immunoglobulin (sIg)+, immunoglobulin gene rearrangement+, ATL‐specific antigen (ATLA)+, HTLV‐1 proviral genome+, Epstein‐Barr virus (EBV)‐associated nuclear antigen (EBNA)+ and the EBV DNA genome+. The fresh T‐leukemic cells were T‐cell receptor gene rearrangement+, the HTLV‐1 proviral genome+ and EBV DNA genome−.

Aberrant expression of the monocyte/macrophage phenotype in a human T cell line immortalized by HTLV‐I and an adult T cell leukemia/lymphoma cell line

An HTLV‐I‐immortalized human T cell line (JP‐2), a N‐methyl‐N′‐nitro‐N‐nitrosoguanidine‐treated JP‐2 line (JP‐2T), and an adult T cell leukemia cell line (ATL‐1T) were examined morphologically and phenotypically. All of these cell lines expressed some T cell markers, including CD4, and showed rearrangement of T cell receptor (TCR) genes, but they lacked CD3 and TCR antigens and expressed some myelomonocytic markers (CD68, HL‐21, CD15, CD16). JP‐2 cells grew in suspension, but JP‐2T and ATL‐1T cells, which mostly adhered to the surface of culture vessels, showed macrophage‐like morphological features and expressed more monocyte/macrophage markers (lysozyme, α1‐antitrypsin) and fibronectin. ATL‐1T cells transplanted into SCID mice lost the macrophage features. These results suggest that HTLV‐I infected T cells can express some macrophage features and that these cells may provide a model that will be useful in elucidating the phenotypic variability of T cell lymphomas.