Use of Encapsulated Stem Cells to Overcome the Bottleneck of Cell Availability for Cell Therapy Approaches.

Nowadays cell-based therapy is rarely in clinical practice because of the limited availability of appropriate cells. To apply cells therapeutically, they must not cause any immune response wherefore mainly autologous cells have been used up to now. The amount of vital cells in patients is limited, and under certain circumstances in highly degenerated tissues no vital cells are left. Moreover, the extraction of these cells is connected with additional surgery; also the expansion in vitro is difficult. Other approaches avoid these problems by using allo-or even xenogenic cells. These cells are more stable concerning their therapeutic behavior and can be produced in stock. To prevent an immune response caused by these cells, cell encapsulation (e.g. with alginate) can be performed. Certain studies showed that encapsulated allo- and xenogenic cells achieve promising results in treatment of several diseases. For such cell therapy approaches, stem cells, particularly mesenchymal stem cells, are an interesting cell source. This review deals on the one hand with the use of encapsulated cells, especially stem cells, in cell therapy and on the other hand with bioreactor systems for the expansion and differentiation of mesenchymal stem cells in reproducible and sufficient amounts for potential clinical use.

Production process for stem cell based therapeutic implants: expansion of the production cell line and cultivation of encapsulated cells.

Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3.

Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias.

Loss of the Y chromosome is a frequent chromosomal imbalance in pancreatic cancer and allows differentiation to chronic pancreatitis.

In a study for the identification of genomic alterations in pancreatic cancer, representational difference analysis was used and led to the isolation of 2 distinct fragments, deleted on the Y chromosome in the xenografted tumor DNA of a male patient with an adenocarcinoma of the pancreas. Loss of Y chromosomal material was further studied in 11 pancreatic cancer cell lines of male origin, using PCR amplification of 5 sequence tagged sites (STSs) distributed along the Y chromosome; 8/11 cell lines exhibited a complete loss of the Y chromosome and 3 had deletions. To examine the status of the Y chromosome in situ, interphase FISH analysis was performed on paraffin sections from pancreatic carcinoma (n=7) and chronic pancreatitis (n=7) tissues, and the loss of Y-chromosomal STS-markers was studied in 6 xenograft tumors obtained from male pancreatic cancer patients. This analysis revealed that a loss of the Y chromosome occurs in vivo in primary pancreatic tumor cells, whereas the Y chromosome was intact in chronic pancreatitis. Our data suggest that loss of Y is a frequent event occurring in male pancreatic tumors. Although there is no evidence for a functional implication of Y chromosome loss, it effectively differentiates between a malignant and a benign condition as e.g. chronic pancreatitis. Thus, this genetic alteration may be of diagnostic use.

A novel transmembrane serine protease (TMPRSS3) overexpressed in pancreatic cancer.

We report the characterization of a novel serine protease of the chymotrypsin family, recently isolated by cDNA-representational difference analysis, as a gene overexpressed in pancreatic cancer. The 2.3-kb mRNA of the gene, named TMPRSS3, is strongly expressed in a subset of pancreatic cancer and various other cancer tissues, and its expression correlates with the metastatic potential of the clonal SUIT-2 pancreatic cancer cell lines. The deduced polypeptide sequence consists of 437 amino acids and exhibits all of the structural features characteristic of serine proteases with trypsin-like activity. TMPRSS3 is membrane bound with a NH2-terminal signal-anchor sequence and a glycosylated extracellular region containing the serine protease domain. Thus, TMPRSS3 is a novel membrane-bound serine protease overexpressed in cancer, which may be of importance for processes involved in metastasis formation and tumor invasion.

Expression of the highly conserved RNA binding protein KOC in embryogenesis.

The human KOC gene which is highly expressed in cancer shows typical structural features of an RNA binding protein. We analyzed the temporal and spatial expression pattern of KOC in mouse embryos at different gestational ages. The expression of KOC seems to be ubiquitous at early stages. During advanced gestation highest KOC expression occurs in the gut, pancreas, kidney, and in the developing brain. The expression pattern of KOC was compared to its Xenopus homologue Vg1-RBP during frog development. Similar expression was found in these organs suggesting an important functional role of the homologous proteins in embryonic development.

Novel technology for detection of genomic and transcriptional alterations in pancreatic cancer.

AIM: The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. METHODS: To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA-RDA (representational difference analysis). Comparative genomic hybridization (CGH) was used to study chromosomal aberrations occurring in pancreatic cancer tissues. RESULTS: The study of transcriptional alterations led to the identification of more than 500 genes with differential expression in pancreatic cancer. The sum of these alterations represented the first expression profile characteristic for pancreatic tumors. The CGH analysis allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. These regions are presently being characterized at the molecular level. In a first approach the myb-oncogene was identified as the relevant oncogene of an amplification on 6q occurring in up to 10% of pancreatic cancer patients. CONCLUSIONS: Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.

Use of representational difference analysis to study the effect of TGFB on the expression profile of a pancreatic cancer cell line.

It has been shown that TGFBs, their receptors, or downstream targets show genetic alterations in pancreatic cancer. This study was designed to identify transcriptional alterations induced by prolonged treatment of pancreatic cancer cell lines with TGFB. The TGFB-responsive PANC-1 cell line was treated with 10-ng/ml TGFB1 for 24 hr. cDNA representational difference analysis was used to generate subtracted hybridization probes enriched for TGFB regulated genes. These probes were hybridized on gridded arrays of cDNA clones containing genes differentially expressed in pancreatic cancer. Twenty-seven distinct cDNA clones were shown to be TGFB target genes. Eleven genes were upregulated by TGFB and were associated with extracellular matrix composition and formation, including genes usually transcribed by cells of mesenchymal origin only. Transcript levels of 16 genes were downregulated by TGFB and could mainly be classified into markers of epithelial differentiation and genes involved in the transcriptional and translational machinery. In conclusion, a 24-hr treatment of PANC-1 cells with TGFB induced a loss of epithelial and a gain of mesenchymal markers. As in other tumors, this epithelial-mesenchymal transdifferentiation may be of general importance during pancreatic carcinogenesis, and may participate, e.g., in the development of the desmoplastic reaction or the acquisition of an invasive phenotype of pancreatic tumor cells. This study demonstrates the usefulness of cDNA RDA and gridded clone libraries to study the effect of signaling cascades on the expression profile of tumor cells. Similar approaches may be helpful in the context of the genome project for the characterization of novel genes. Genes Chromosomes Cancer 26:70-79, 1999.

Strategies for the detection of disease genes in pancreatic cancer.

The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA RDA (representational difference analysis). These approaches have led to the identification of more than 500 genes with differential expression in pancreatic cancer. To study chromosomal aberrations occurring in pancreatic cancer tissues we used comparative genomic hybridization (CGH). This allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.

Isolation of differentially expressed genes by combining representational difference analysis (RDA) and cDNA library arrays.

The difference products (DP) of representational difference analyses (RDA) were used as hybridization probes on cDNA arrays. The effectivity of RDA products obtained with increasing driver/tester ratios (DP 1 = 100:1, DP 2 = 800:1 and DP 3 = 400,000:1) to isolate differentially expressed genes was compared with the effectivity of conventional differential hybridizations. Pacreatic cancer and control tissues were used as a test system to isolate differentially expressed genes. The use of RDA products as hybridization probes showed two major advantages: (i) a reliable identification of true differential signals; and (ii) only one autoradiograph had to be analyzed, which eliminated the need for a laborious subtraction of signal intensities obtained with different cDNA probes. Increasing driver/tester ratios in iterative rounds of RDA delivered more specific results, though the total yield of differential clones was gradually reduced. In this situation, the intermediate RDA product DP 2 provided the best compromise.

Identification of a new tumour-associated antigen TM4SF5 and its expression in human cancer.

In a previous large-scale screening for differentially expressed genes in pancreatic cancer, a gene was identified that was highly overexpressed in pancreatic cancer encoding a novel putative tetraspan transmembrane protein highly homologous to the tumour-associated antigen L6. Using a radiation hybrid panel the identified human gene named TM4SF5 (transmembrane 4 superfamily member 5) was localized to chromosome 17 in the region 17p13.3. The cloned TM4SF5 cDNA has a 32bp 5'-untranslated region (UTR), a 591bp open reading frame (ORF) and a 85bp 3'UTR. The predicted TM4SF5 protein with 197 amino acids contains three NH2-terminal hydrophobic transmembrane regions, followed by an extracellular hydrophilic domain containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. These structural features are shared by the L6 antigen and a number of related cell surface proteins associated with cell growth. TM4SF5 was overexpressed in pancreatic cancer tissues as compared to both normal pancreas and chronic pancreatitis tissues, and was detected at high levels in other tumour tissues. Although the precise function of TM4SF5 remains to be elucidated it may be useful in a clinical setting for tumour diagnosis and/or therapy. This hypothesis is supported by the strong homology to the L6 antigen, which has proved promising in immunological, therapeutic and diagnostic approaches.

Cloning of a new Kunitz-type protease inhibitor with a putative transmembrane domain overexpressed in pancreatic cancer.

In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel putative transmembrane protein with two Kunitz-type serine protease inhibitor domains. The identified gene named kop (Kunitz domain containing protein overexpressed in pancreatic cancer) was assigned to chromosome 19 in the region 19q13.1. Kop was detected at high levels in pancreatic cancer cell lines and was overexpressed in pancreatic cancer tissues as compared to both, normal pancreas and chronic pancreatitis tissues. Being a member of the Kunitz-type serine protease inhibitor family, this new gene may participate in tumour cell invasion and metastasis and in the development of the marked desmoplastic reaction typical for human pancreatic cancer tissues. In this context, the fact that kop has a putative transmembrane domain may have functional implications of particular interest.

The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity.

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.

Characterization of a high copy number amplification at 6q24 in pancreatic cancer identifies c-myb as a candidate oncogene.

In a recent study designed to identify chromosomal aberrations in pancreatic cancer tissues using comparative genomic hybridization, a high copy number amplification on 6q was detected. To identify the most likely candidate oncogene, the extension of the amplification in pancreatic cancer tissues and cell lines was determined by Southern blot analysis. Exon trapping was performed with DNA from a yeast artificial chromosome clone containing the complete minimally amplified region. Only fragments from two genes, namely, the c-myb oncogene and a novel gene, were shown to be amplified. The c-myb proto-oncogene was amplified in 10% of the pancreatic carcinoma tissues and in the pancreatic cancer cell line PC2. Interestingly, the c-myb oncogene was overexpressed not only in the amplified samples but also in the majority of the examined pancreatic cancer tissues and cell lines, suggesting that amplification is only one of the mechanisms leading to overexpression. In contrast, the novel gene, which was called human eRF3b (eukaryotic release factor 3b), seems to be only coamplified with c-myb. Genetic alterations of c-myb were mainly found in advanced tumors, indicating a possible correlation to tumor progression and aggressive tumor phenotypes.

Cloning of a gene highly overexpressed in cancer coding for a novel KH-domain containing protein.

In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel protein with four K-homologous (KH) domains. KH-domains are found in a subset of RNA-binding proteins, including pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). By fluorescence in situ hybridization (FISH) the identified gene named koc (KH domain containing protein overexpressed in cancer) was assigned to chromosome 7p11.5. Two pseudogenes were localised on chromosome 6 and 11. The cloned koc cDNA has a 250 bp 5'-UTR, a 1740 bp ORF and a 2168 bp 3'-UTR. The AU-rich 3'-untranslated region of koc contains eight AUUUA and four AUUUUUA reiterated motifs. The deduced koc protein with 580 amino-acids has a relative molecular mass (Mr) of approximately 65,000 (65 K). The koc transcript is highly overexpressed in pancreatic cancer cell lines and in pancreatic cancer tissue as compared to both, normal pancreas and chronic pancreatitis tissue. High levels of expression were as well found in tissue samples of other human tumours. As the KH domain has been shown to be involved in the regulation of RNA synthesis and metabolism, we speculate that koc may assume a role in the regulation of tumour cell proliferation by interfering with transcriptional and or posttranscriptional processes. However, the precise role of koc in human tumour cells is unknown and remains to be elucidated.

Identification of genes with specific expression in pancreatic cancer by cDNA representational difference analysis.

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.

Mapping of chromosomal imbalances in pancreatic carcinoma by comparative genomic hybridization.

To identify recurrent chromosomal imbalances in pancreatic adenocarcinoma, 27 tumors were analyzed by using comparative genomic hybridization. In 23 cases chromosomal imbalances were found. Gains of chromosomal material were much more frequent than losses. The most common overrepresentations were observed on chromosomes 16p (eight cases), 20q (seven cases), 22q (six cases), and 17q (five cases) and under-representations on a subregion of chromosome 9p (eight cases). Distinct high-level amplifications were found on 1p32-p34, 6q24, 7q22, 12p13, and 22q. These data provide evidence for a number of new cytogenetically defined recurrent aberrations which are characteristic of pancreatic carcinoma. The overrepresented or underrepresented chromosomal regions represent candidate regions for potential oncogenes and tumor suppressor genes, respectively, possibly involved in pancreatic tumorigenesis.