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Mesenchymal stem cells (MSCs) have recently made significant progression in multiple clinical trials targeting several clinical disorders and in the modulation of immune responses. In the present study, we isolated human adipose tissue-derived stem cells (ADSCs) by direct membrane migration method without using enzymatic digestion via collagenase, and tried to extract adequate number of cells for clinical application. Hydroxyapatite-treated nonwoven fabric membrane made up of synthetic macromolecular fiber materials, polyethylene and polyester terephthalene was used. Expansion culture of ADSCs having plastic flask adherent characteristic in serum-free condition was successfully established, and adequate number of cells were obtained for clinical application. They were found to be positive for CD44, CD73, CD90 and CD105 and negative for CD11b, CD34, CD45, CD80 and HLA-DR. The resulting immunological marker profile satisfied the immunophenotype of previously reported MSCs. Also, microscopic findings demonstrated trilineage differentiation into adipogenic, osteogenic and chondrogenic cells as the characteristics of MSCs. The isolation by nonwoven fabric membrane and expanded cells under serum-free condition satisfied the criteria of MSCs, as proposed by the International Society for Cellular Therapy. Our direct membrane migration method without enzyme digestion is useful as ADSCs can be obtained from small pieces of adipose tissue and expanded under serum-free culture condition. This method was considered to be feasible for clinical application.

Although a number of transcription factors (TFs) have been identified that play a pivotal role in the development of hematopoietic lineages, only little is known about factors that may influence development and lineage commitment of natural killer (NK) or NK-like T (NKT)-cells. Obviously to fully appreciate the NK- and NKT-cell differentiation process, it is important to identify and characterize the TFs effecting the NK- and NKT-cell lineage. Furthermore, these TFs may play a role in NK- or NKT-cell leukemias, in which the normal differentiation program is presumably disturbed. The present study analyzed the expression of the following 13 TFs: AML1, CEBPA, E2A, ETS1, GATA1, GATA2, GATA3, IKAROS, IRF1, PAX5, PU1, TBET and TCF1 in 7 malignant NK-cell lines together with 5 malignant NKT-cell lines, 5 T-cell acute lymphoblastic leukemia (ALL) cell lines including 3 gamma/delta T-cell receptor (TCR) type and 2 alpha/beta TCR type, and 3 B-cell precursor (BCP) leukemia cell lines. AML1, E2A, ETS1, IKAROS and IRF1 were found to be positive for all cell lines tested whereas GATA1 turned out to be universally negative. CEBPA, PAX5 and PU1 were negative for all cell lines tested except in the three positive BCP-cell lines. GATA2 was positive for 3/5 T-cell lines but negative for the other cell lines. GATA3 was positive for 7/7 NK-, 4/5 NKT-, 5/5 T- and 2/3 BCP-cell lines. TBET was positive for all NK- and NKT-cell lines and negative for all T- and BCP-cell lines except one BCP-cell line. In contrast to the expression of TBET, TCF1 was negative for all NK- and NKT-cell lines, being positive for 4/5 T- and 1/3 BCP-cell lines. Expression analysis of TFs revealed that NK- and NKT-cell lines showed identical profiles, clearly distinct from those of the other T-ALL or BCP-ALL leukemia-derived cell lines..

Multiple myeloma (MM) is a neoplasm of a terminally differentiated B-cell. Human myeloma cell lines were shown to be suitable model systems for use in various fields of the biological sciences. Within the last 20 years more than 100 cell lines have been established. So-called 'myeloma cell lines' have been previously reported and are still widely used which are in reality Epstein-Barr virus (EBV)-positive B-lymphoblastoid cell lines. The presence of the EBV-genome in residual normal B-cells provides them with a selective growth advantage after explantation. Cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. On closer examination, the use of false cell lines may be seen to invalidate a significant percentage of scientific work, or at least cast doubts on the relevance of these in vitro results to the cell type or tumor in vivo. Ultimately, use of cross-contaminated cell lines is a waste of human and material resources. Henceforth, it should be mandatory to prove the proper derivation of each new cell line by comparing DNA fingerprints or karyotypes of the patient's primary cells and the cultured cells. The availability of well characterized and authenticated bona fide MM cell lines is of great importance for the study of the biology, etiology and treatment of the disease.

A novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) cell line, NALM-35, was established from the peripheral blood of a 40-year-old woman at diagnosis of ALL. Imunophenotyping showed BCP type III characteristics including expression of TdT, CD10, CD19, CD22, CD79a and HLA class II. T-cell and myeloid-associated antigens tested were negative except CD5 and CD28. The surrogate light chains CD179a and CD179b were positive. NALM-35 cells have the morphological appearance of lymphoblasts. Cytogenetic analysis of NALM-35 revealed an abnormal karyotype with 46, XX, add(9)(p11). Southern blot analysis of the immunoglobulin genes status of NALM-35 at 10 months after establishment showed germ line configuration of the kappa and lambda light chain genes, and rearrangement of the mu heavy chain gene. DNA fingerprinting, chromosomal analysis and immunophenotyping proved that NALM-35 was clonally derived from the primary leukemia cells. The established cell line may provide a useful model system and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.

A series of human acute lymphoblastic leukemia (ALL) cell lines, BALM-19, -20, -21, -22, -23 (BALM 19-23) and BALM-26 were established from a patient with B-cell characteristics of ALL L2 type. All cell lines were derived from bone marrow specimens, BALM 19-23 from a sample taken at diagnosisand BALM-26 from one at relapse. Like the original leukemia cells, the established lines present various B-cell characteristics, being positive for cell surface immunoglobulin (Ig) chains but also for nuclear terminal deoxynucleotidyl transferase; hence the cell lines should be assigned to B-cell category B-IV. As a unique feature, the cell lines expressed the CD33 myeloid antigen in addition to the common B-cell markers. Heterogeneous antigen expression among the different cell lines was found regarding CD35, CD39, CD45RA, CD78 and CD95. The malignant nature of the cell lines was documented by negativity for the Epstein-Barr virus and by the occurrence of clonal non-random structural chromosome abnormalities. The patient's serum showed hypercalcemia, prompting further investigation of the established cell lines which expressed parathyroid hormone related peptide (PTHrP) mRNA as examined by reverse transcriptase polymerase chain reaction. The established B-cell ALL sister cell lines, BALM 19-23 and BALM-26, could provide useful material for clarifying the pathogenesis of this type of B-cell malignancy. The scientific significance of this panel of cell lines lies in the availability of a series of clonally derived but phenotypically different sister cell lines established at different phases of the disease.

Human bone marrow stroma (BST)-dependent myeloma sister cell lines MOLP-6 and MOLP-7 were established from the peripheral blood of a multiple myeloma (MM) patient with IgA kappa type MM (stage IIIB). The growth of the cell lines is constitutively dependent on BST cells; none of the cytokines tested nor the culture supernatant of the BST cells could support the growth. Both cell lines showed typical plasma cell morphology with abundant cytoplasm and one to four nuclei under Wright staining. The immunoprofiles of MOLP-6 and MOLP-7 correspond to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) chains, a heavy and kappa light chains, CD9, CD28, CD40, CD44, CD45, CD56, and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte associated markers. Both cell lines also expressed adhesion molecules including HCAM (CD44), VLA-4 (CD49d/CD29), VLA-6 (CD49f/CD29), ICAM-1 (CD54), NCAM (CD56), LFA-3 (CD58) and L-selectin (CD62L). The doubling time of MOLP-6 and MOLP-7 was 48 and 168 hours, respectively. In addition to this growth characteristic, the maximum cell density of each cell line was obtained at 1.7 x 10(6) cells/ml and 9.7 x 10(5) cells/ml, respectively. The characteristics of each cell line may reflect intraclonal variation of the proliferative capacity. The MOLP-6 together with the MOLP-7 sister will be useful model systems for the investigation of the biology of myeloma.

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

We established three sister cell lines, NALM-30, NALM-31 and NALM-32, with biphenotypic features carrying myeloperoxidase mRNA and protein with complex Philadelphia (Ph) chromosome, t(9;22;10)(q34;q11;q22), from a patient with Ph-positive acute leukemia in relapse. Epstein-Barr virus nuclear antigen was negative. The morphological appearance of the cell lines is that of immature lymphoid cells. Expression of myeloid- and lymphoid-associated surface membrane antigens on these cells was detected allowing for the classification of "biphenotypic" leukemia. Immunophenotypically, the established cell lines reported here fulfill the European Group for the Immunological Characterization of Leukemias (EGIL) criteria for B-lineage derivation, however, surface and cytoplasmic immunoglobulin chains were negative. Whereas TGF-beta R (CD105), MCSFR (CD115), SCFR (CD117), IL-4R/IL-13R (CD124) and IL-6R (CD126) were not expressed, the cell lines were mostly positive for IFN-gamma R (CD119), IL-7R (CD127) and FLT-3R (CD135). The NALM-30, NALM-31 and NALM-32 cell lines together with their serial sister cell lines NALM-27 and NALM-28 which were established from the same patient at diagnosis provide unprecedented opportunities for studying a multitude of biological aspects related to normal and neoplastic immature B-lymphocytes.

Continuous human leukemia-lymphoma cell lines have become invaluable tools for hematological research as they provide an unlimited amount of cellular material. The first human lymphoma cell line Raji was established in 1963; since then several hundred leukemia-lymphoma cell lines spanning almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) have been described. The cardinal features of leukemia-lymphoma cell lines are their monoclonal origin, arrest of differentiation, and (growth factor-independent or -dependent) unlimited proliferation. Categorization of cell lines usually follows the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, a detailed characterization of both primary and cultured cells in absolutely necessary. New cell lines, in particular, must be adequately, characterized; while cell culture data and immunological and cytogenetic features are essential, cell lines should be described in as much detail as possible. In addition to this recommended multiparameter characterization and the obligatory immortality of the culture, authentication of the true origin of the cells, novelty, scientific significance and availability of the cell line for other investigators are of utmost importance. It is still extremely difficult to establish new leukemia-lymphoma cell lines (except for some subtypes), and most attempts fail. Paramount to the lack of our understanding as to why certain cells start to proliferate in culture and others do not (thus implying a random process), is probably the difficulty of mimicking in vitro the physiological in vivo microenvironment. Attempts to improve the efficiency of cell line establishment should focus on examining the appropriateness of the in vitro culture conditions; these conditions should emulate as closely as possible the in vivo situation. In summary, leukemia-lymphoma cell lines have the potential to greatly facilitate diverse studies of normal and malignant hematopoiesis; to that end, these cell lines must be extensively characterized and adequately described.

We established two novel PH-positive acute leukemia cell lines with "biphenotypic" feature, NALM-27 and NALM-28, with t(9;22;10)(q34;q11;q22) from a patient with biphenotypic acute leukemia(BAL). The breakpoint cluster region (bcr) of the BCR gene was found to be rearranged in these cells by Southern blot analysis using a major 3'-side bcr probe. Polymerase chain reaction (PCR) results showed differences in the pattern of expression of the abl-bcr fusion gene in comparison with the diagnosis. In the case of variant Ph translocations, reports have appeared concerning mainly chronic myelogenous leukemia (CML), but there have been few concerning acute leukemia with lymphoid feature. This study thus identifies nonrandomly involved chromosome sites which can then be targeted for detailed molecular analysis to obtain an understanding of abl-bcr fusion in the cells with lymphoid feature. In addition, chromosome band 10q22, involved in this translocation, is the site for several neoplasia. Furthermore, this site is non-randomly involved in the formation of variant Ph translocations in acute lymphoblastic leukemia (ALL). This is the first report on the t(9;22;10)(q34;q11;q22) rearrangement in NALM-27 and NALM-28 cell lines which should prove useful for understanding the translocation of molecular breaks within the bcr of the complex translocation site.

The human Evi-1 gene located on chromosome 3q26, encodes a zinc finger protein that functions as a transcription factor. It was frequently overexpressed in leukemias having 3q26 abnormalities such as t(3;3)(q21;q26) and inv(3)(q21 q26), and subjected to structural alteration in t(3;21)(q26;q22). In addition, recent studies indicated that several cases of leukemias without 3q26 abnormalities also expressed Evi-1 gene. In this study we present another case of structural alteration of Evi-1 gene in a case of inv(3)(q21 q26), in which Evi-1 was truncated and a shorter form of Evi-1 protein was expressed upon rearrangement of the gene. We also studied expression of the Evi-1 gene in a variety of leukemias by northern blot analysis. Evi-1 was overexpressed not only in leukemias with 3q26 abnormalities, but, in those without 3q26 abnormalities, especially in blast crisis of CML. Our result also supports an idea that Evi-1 is a relevant oncogene whose overexpression or structural changes might play a crucial role in development of human leukemias.

Two new B-cell lines, BALM-13 and BALM-14, were established from the bone marrow aspirate of a 13-year-old male patient with acute leukemia. These cell lines are unique in their expression of CD antigens. BALM-13 was characterized as belonging to the Burkitt lymphoma group III cell type (CD10-, CD20+, CD23+, D39+, CD77-), and BALM-14 to the Burkitt lymphoma group I cell type (CD10+, CD20+, CD23-, CD39-, CD77+). The expression of immunoglobulin chains of BALM-13 (lambda delta mu) differed from those of BALM-14 (lambda mu). Furthermore, BALM-13 was positive for Epstein-Barr virus nuclear antigen but BALM-14 was negative. This is a unique pair of cell lines having intraclonal phenotypic heterogeneity.

A new pre-B cell leukemia cell line, NALM-26, was established from the peripheral blood of a 24-year-old male patient with acute pre-B cell leukemia. NALM-26 is unique in its expression of T cell-associated CD5 and myeloid cell-associated CD13 antigens. Interleukin-7 (IL-7) receptor (CDw127) was detected by flow cytometric analysis. After PMA treatment, NALM-26 was induced to express CD20, CD25 and CD28, and to increase its expression of both CD5 and CD13. The expression of CDw127 was down-modulated.

Seven cell lines, MOLM-6, -7, -8, -9, -10, -11 and -12, were established from a single blood sample from a patient with chronic myelogenous leukemia (CML) in blastic phase having the Ph1 chromosome abnormality. Based on immunophenotyping, two of these seven cell lines, MOLM-7 and -11, represented the megakaryoblastoid lineage, and the other five cell lines represented two different maturation stages of the myeloid lineage.

Multiple myeloma cell lines, MOLP-2 and MOLP-3, were established from the peripheral blood of a patient in leukemic phase of multiple myeloma. Both cell lines express the IL-6 receptor, CD28 T cell-associated antigen and CD33 myeloid-associated antigen. IgD lambda immunoglobulin was found in the culture supernatants of both MOLP-2 and MOLP-3.

Three leukemia cell lines, BALM-6, -7 and -8 were established from the bone marrow of a patient with acute lymphoblastic leukemia. Based on immunophenotypic and the cytogenetic analysis and on the expression of immunoglobulins on both the cell surface and in the cytoplasm, BALM-6, BALM-7 and BALM-8 are clonal leukemic cell populations of B cell origin.

Four leukemia cell lines; NALM-20, established at the onset of leukemia and NALM-21, -22 and -23 established at the relapse of the disease were found to be t(9;22)-positive leukemia lines having the biphenotypic feature of B cell and myeloid cell characteristics. In addition, a polyclonal Epstein-Barr virus-transformed normal B cell line, B250, was established from the peripheral blood at the onset of the disease.

Based on the immunophenotypic, cytogenetic and genotypic findings, two unique leukemia cell lines, NALM-24 and NALM-25, and an EBV-transformed "normal" B-lymphoblastoid cell line (B262) from a patient with ALL were established and characterized. NALM-24 and NALM-25 are unique in that expression of both show B cell and myeloid cell features with the t(9;22) chromosome in single clonal leukemic cell populations.

Based on the immunophenotypic and genotypic findings, this acute leukemia cell line, designated NALM-19, is unique in that a partial expression of both B-cell and myeloid cell features are present in this single clonal leukemic cell population. It is noteworthy that two "normal" EB virus-transformed B cell lines, B239 and B240, (paired with NALM-19) were established from the same leukemic blood.

Based on the morphological resemblance to megakaryocytes and upon the expression f the platelet specific glycoprotein antigen, CD61, it is highly l y that MOLM-1 is a clonal leukemic cell population of megakaryoblastic origin .

A total of 33 human leukemia/lymphoma cell lines were classified into 4 groups with respect to the pattern of cell membrane (sm) expression of the CD3 and T cell receptor (TCR) molecules; (i) smCD3+TCR alpha beta (16 cell lines), (ii) smCD3+TCR beta delta (1 cell line), (iii) smCD3+TCR gamma delta (3 cell lines) amd (iv) smCD3-TCR- (13 cell lines), respectively. Using monoclonal antibodies (MoAbs) specific to CD3 (NU-T3), TCR alpha chain (alpha F1), TCR beta chain (beta F1), and TCR gamma chain (C gamma M1), respectively, cytoplasmic (cy) expression of these molecules was determined by immunofluorescence test. Expression of cyCD3 was present in all cell lines regardless of groups. In group (i), all 16 cell lines expressed both TCR alpha and beta chains. While only TCR beta chain was expressed in group (ii), TCR gamma chain was expressed in all 3 cell lines of group (iii). One (PEER) of the three in group (iii) expressed TCR beta chain as well. In group (iv), we found 8 cell lines with cyTCR alpha expression, 11 cell lines with cyTCR beta expression, and 10 cell lines with cyTCR gamma expression, respectively. For TCR genes, except 1 cell line all cell lines were found to present rearranged C beta gene and its mRNA, including all 3 TCR gamma/delta cell lines of group (iii). One of the TCR alpha beta cell lines exhibited rearranged C delta and J delta genes as well as its mRNA. Two cell lines of the 13 CD3-TCR- of group (iv) exhibited rearranged C delta and J delta and its mRNA. An NK-like activity and IL-2 production were induced in the TCR beta delta and gamma delta cell lines [group (ii) and (iii)] by treatment with PHA and PMA.

Gastrointestinal hormones have been used for diagnostic and therapeutic applications. Although it is assumed that gastrointestinal hormones may be involved in many diseases of the gut. So far, however, we can identify significant roles only in peptic ulcer disease, syndromes caused by gastrointestinal hormone-producing tumors, chronic pancreatitis, and sprue. Future investigation will make the role of gastrointestinal hormone clearer in many diseased states.

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.

Establishment and total characterization of permanent growth-factor independent human leukemia cell lines are reviewed. Among others, a few significant contributions in the utility of leukemia cell lines described include establishment of immunodiagnosis of human leukemias and lymphomas, discovery of EB virus and of human retroviruses, proposed model of normal hematopoietic cell differentiation scheme, lymphokine-monokine production, identification and characterization, studies on genes responsible for immunobiological and growth regulatory molecules, and pharmacotoxicological and kinetic research. It is conceivable that the future leads by the utility of human leukemia cell lines to the advancement of research are indeed unlimited.