Hossein Baharvand

Klaus Ingo Matthaei

Establishment of New Embryonic Stem Cell lines

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. These cells are appropriate for creation of animal models of human genetic diseases, the study of gene function in vivo and differentiation into specific types as potential therapeutic agents for several human diseases. We describe here the production of new ES cell lines from blastocysts recovered from the C57BL/6 and BALB/c mouse strains by changing in concentration of Leukemia inhibitory factor (LIF) and primary culture conditions. The established cell lines were analyzed by simple karyotype, C-banding, Alkaline phosphatase activity, OCT-4 expression as well as for the presence of the SRY gene. Two ES cell lines from C57BL/6 and three from the BALB/c were produced. The two C57BL/6 ES cell lines were established with either 1000 or 5000 i.u. LIF whereas the BALB/c ES cell lines required 5000 i.u. LIF. Four of the ES cell lines had a normal karyotype. C-banding and SRY-PCR showed that all cell lines had an XY sex chromosome composition. All five of the cell lines expressed alkaline phosphatase activity and OCT-4. One of the BALB/c ES cell lines when injected into C57BL/6 blastocysts produced high rates of chimerism as assessed by coat color and the male chimera produced germline offspring when mated with BALB/c females. These results indicate that ES cells from inbred strains can be isolated using commercially available reagents and that the establishment of BALB/c ES cell lines may require different culture conditions to the 129 or C57BL/6 strains. Hossein Baharvand and Klaus Ingo Matthaei Culture Condition Difference for Establishment of New Embryonic Stem Cell Lines from the C57BL/6 and BALB/c Mouse Strains. In Vitro Cellular and Developmental Biology – Animal, 40:76-81, 2004.


Brassica Biotechnology: Progress in Cellular and Molecular Biology

Considerable progress has been accomplished in the cellular and molecular biology of Brassica species in the past few years. Plant regeneration has been increasingly optimized via organogenesis and somatic embryogenesis using various explants; tissue culture improvements focusing on factors such as age of the explant, genotype, and media additives. The production of haploids and doubled haploids using microspores has accelerated the production of homozygous lines in the Brassica species. Somatic cell fusion has facilitated the development of interspecific and intergeneric hybrids in the sexually incompatible species of Brassica. Crop improvement using somaclonal variation has also been achieved. The use of molecular markers in marker assisted selection and breeding, transformation technology for the introduction of desirable traits and a comparative analysis of these as well as their future prospects are important parts of current research that are reviewed. Vinitha Cardoza and C. Neal Stewart, Jr. Brassica Biotechnology: Progress in Cellular and Molecular Biology, In Vitro Cellular and Developmental Biology – Animal, 40:542-551, 2004.


Hybrid Cell Line Generation

We have previously isolated mouse embryonic cell lines with endothelial potential using a simple empirical approach. In an attempt to isolate similar cell lines from adult mouse bone marrow (BM), BM cells were cultured on mitotically inactive mouse embryonic fibroblasts (MEFs) feeder. Several cell lines with putative endothelial potential were generated. They expressed endothelial specific genes and formed vascular-like structures when plated on matrigel. When transplanted into appropriate mouse models, they incorporated into the endothelium of the vasculature. Similar cell lines were also obtained using human or porcine BM. None of these lines induced tumor formation when transplanted into immunodeficient Rag1-/- mice. However, all the lines were aneuploid with genetic markers from BM samples and the MEF feeder suggesting that they resulted from a non-species specific fusion of a BM cell and mitotically inactive MEF. Together, these lines demonstrated for the first time that BM cells can also undergo fusion with commonly used mitotically inactive feeder cells. Although these fusion cell lines were culture artifacts, their derivation would be useful in understanding fusion of BM cells with other cell types and their endothelial potential will also be useful in characterizing endothelial differentiation. Jianwen Que, Reida Menshawe El Oakley, Manuel Salto-Tellez, Dominque P. V. de Kleijn, Ming Teh, Leslie Retnam, and Sai-Kiang Lim. Generation of Hybrid Cell Lines with Endothelial Potential from Spontaneous Fusion of Adult Bone Marrow Cells with Embryonic Fibroblast Feeder, In Vitro Cellular and Developmental Biology – Animal, 40:143-149, 2004.


Safedin Beqaj, Jon M. Ryan, and Anuja Shah

Kidney Cells Responsible for Urokinase Synthesis and Secretion

Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium. Safedin Beqaj, Anuja Shah and Jon M. Ryan. Identification of Cells Responsible for Urokinase-type Plasminogen Activator Synthesis and Secretion in Human Diploid Kidney Cell Cultures, In Vitro Cellular and Developmental Biology – Animal, 40:102-107, 2004.


 

Kirsten H. Walen

Spontaneous Cell Transformation

Previously it was shown that SV40-induced cell transformation of human diploid (2N), epithelial cells, was a dynamic process of nuclear and cellular events. In this process nuclei of polyploid (above 2N) cells broke-down into multinucleated cells (MNCs) by amitotic division. An induced mass karyoplast (ie., small cell with reduced amount of cytoplasm) budding process from the MNCs produced transformed cells with extended lifespan (EL) and altered morphology. In the present study without the use of SV40 and no induction of karyoplast budding, the same sequence of cellular events was found to occur spontaneously for the same type of cells at replicative senescence (no mitosis). These cell transformation events were followed by phase contrast photography of living cell cultures. Primary diploid, epithelial cell cultures grew for 2 to 3 passages and then entered senescence. Cells remaining in the cultures after widespread cell death (mortality stage 1; M1) developed the typical large, flat-cell morphology of senescence with increased cytoplasmic volume. Some of these cells were MNCs mostly with 2 to 4 nuclei. Cytokinesis in MNCs and spontaneous karyoplast budding from MNCs were observed and new, limited EL cell growth was present either in foci of cells or as prolonged cell growth over 1 to 2 passages. At the end of their replicative phase the EL cells entered another death crisis (M2) from which no cells survived. In M2-crisis rarely transformed cells appear with immortal cell growth characteristics (ie., cell lines). Numerous examples of fragmentation/amitosis of polyploid nuclei in the production of MNC are presented. Such nuclear divisions produced nuclei with unequal sizes, which suggest unbalanced chromosomal segregations. The nuclear and cellular events in cell transformation is compared to a natural (no induction) occurrence of MNC-offspring cells in mammalian placentas. The possibility of a connection between these two processes is discussed. And, finally the difference in duration of EL cell growth from SV40-MNCs versus from senescent-MNCs is ascribed to increased mutational load in SV40-induced MNCs as compared to that in senescence MNCs. Kirsten H. Walen. Spontaneous Cell Transformation: Karyoplasts Derived from Multinucleated Cells Produce New Cell Growth in Senescent Human Epithelial Cell Cultures, In Vitro Cellular and Developmental Biology – Animal, 40:150-158, 2004.


Effects of MeJA on Cell Growth and Secondary Metabolism

Methyl jasmonate (MeJA) interacted significantly with both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA) to influence cell growth of cultured Onosma paniculatum cells. Cell growth decreased with increasing concentrations of MeJA from 0.004-4.45 ìM with or without IAA and BA. The same concentrations of MeJA (0-4.45 ìM) increased the cell growth with IAA and BA, when administered to the cultured cells in M9 medium. This was found to enhance the production of shikonin. The optimum time for MeJA addition for enhanced shikonin formation was 4 d after cell inoculation in M9 medium. Furthermore, shikonin formation was affected significantly by both MeJA/IAA and MeJA/BA combinations. Shikonin content was enhanced by increasing MeJA concentrations with IAA concentrations in the range of 0-28 ìM and with BA concentrations in the range of 0-44.38 ìM in MeJA/IAA and MeJA/BA experiments, respectively. The optimal combination of MeJA and IAA was 4.45 ìM and 0.28 ìM, while MeJA and BA concentrations of 4.45 ìM and 2.22 ìM were optimal for shikonin formation. The result also showed that MeJA increased phenylalanine ammonialyase (PAL) and phydroxybenzoic acid geranyltransferase (PHBgeranyltransferase) activities during the course of shikonin formation, but decreased the activity of PHBOglucosyltransferase within 9 d after inoculation. These results suggest that enhanced shikonin formation in cultured Onosma paniculatum cells induced by MeJA involves regulation of the key enzyme activities. Jian Ding, Shuai Shie, Bao-Hua Jiang, Yong-Hua Yang, Jie Huan, Heng-Guan Shen, Kai Xia, Junfeng Zhang, and Xiqun Jiang. Effects of Methyl Jasmonsate with Indole-3-Acetic Acid and 6-Benzylaminopurine on the Secondary Metabolism of Cultured Onosma paniculatum Cells, In Vitro Cellular and Developmental Biology – Plant, 40:581-586, 2004.