Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line.

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP).

BACKGROUND: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair. METHODS: The testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC). RESULTS: No human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin. CONCLUSIONS: In summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.

Two cases of atypical meningioma with pulmonary metastases: a comparative cytogenetic analysis of chromosomes 1p and 22 and a review of the literature.

We present two cases of atypical meningioma WHO grade II with a history of multiple local recurrences and late pulmonary metastases. Comparative cytogenetic analyses on 1p and 22q confirmed clonal origin of the primary intracranial meningiomas and the pulmonary metastases in both cases. These cases illustrate the importance of close neuroradiological follow-up to detect tumor recurrence in patients with atypical meningiomas WHO grade II even with clinically stable disease and should sensitize clinicians to late extracranial metastases of these tumors, especially to the lung. In an effort to elucidate common clinical features of metastatic meningiomas, especially to the lung, the literature was reviewed from 1995 to 2014, identifying a total of 45 published cases.

Three gangliogliomas: results of GTG-banding, SKY, genome-wide high resolution SNP-array, gene expression and review of the literature.

According to the World Health Organization gangliogliomas are classified as well-differentiated and slowly growing neuroepithelial tumors, composed of neoplastic mature ganglion and glial cells. It is the most frequent tumor entity observed in patients with long-term epilepsy. Comprehensive cytogenetic and molecular cytogenetic data including high-resolution genomic profiling (single nucleotide polymorphism (SNP)-array) of gangliogliomas are scarce but necessary for a better oncological understanding of this tumor entity. For a detailed characterization at the single cell and cell population levels, we analyzed genomic alterations of three gangliogliomas using trypsin-Giemsa banding (GTG-banding) and by spectral karyotyping (SKY) in combination with SNP-array and gene expression array experiments. By GTG and SKY, we could confirm frequently detected chromosomal aberrations (losses within chromosomes 10, 13 and 22; gains within chromosomes 5, 7, 8 and 12), and identify so far unknown genetic aberrations like the unbalanced non-reciprocal translocation t(1;18)(q21;q21). Interestingly, we report on the second so far detected ganglioglioma with ring chromosome 1. Analyses of SNP-array data from two of the tumors and respective germline DNA (peripheral blood) identified few small gains and losses and a number of copy-neutral regions with loss of heterozygosity (LOH) in germline and in tumor tissue. In comparison to germline DNA, tumor tissues did not show substantial regions with significant loss or gain or with newly developed LOH. Gene expression analyses of tumor-specific genes revealed similarities in the profile of the analyzed samples regarding different relevant pathways. Taken together, we describe overlapping but also distinct and novel genetic aberrations of three gangliogliomas.

High-resolution genomic profiling and locus-specific FISH in subcutaneous and visceral adipose tissue of obese patients.

Obesity is known as a heterogeneous and multifactorial disease. The distribution of body fat is crucial for the development of metabolic complications. Comprehensive genetic analyses on different fat tissues are rare but necessary to provide more detailed information. Therefore, we performed genetic analyses of three patients with obesity using high resolution genome wide SNP array (blood, visceral fat tissue) and fluorescence in situ hybridization (FISH) analyses (visceral and subcutaneous fat tissue). Altogether, we identified 31 small Copy Number Variations (losses: 1p31.1, 1p22.2, 1q21.3, 2q34, 2q37.1, 3q28, 6p25.3, 7q31.33, 7q33, 8p23.3, 10q22.3, 11p15.4, 11p15.1, 11p14.2, 11p12, 13q12.3, 15q11.2-q13.1, 15q13.3, 20q13.2, 22q11.21; gains: 2q22.1-q22.2, 3p14.3, 4p16.3, 4q32.2, 6q27, 7p14.3, 7q34, 11p12, 12p11.21, 16p11.2-p11.1, 17q21.31) and 289 small copy-neutral Loss of Heterozygosity (cn-LOH). For the chromosomal region 15q11.2-q13.1, we detected a microdeletion (Prader-Willi-Syndrome) in one patient. Interestingly, we identified chromosomal SNP differences between EDTA-blood and visceral fat tissue (deletion and gain). Small losses of 7q31.33, 7q33, 11p14.2, 11p12, 13q12.3 as well as small gain of 7q34 were detected only in fat tissue and not in blood. Furthermore, FISH analyses on 7q31.33, 7q33 and 11p12 revealed differences between subcutaneous and visceral fat tissue. Generally, the deletions were detected more frequent in visceral fat tissue. Predominantly detected cn-LOH vs. CNV suggests a meaning of these cn-LOH for the pathogenesis of obesity. We conclude that the SNP array and FISH analyses used is applicable to generate more information for basic research on difficult cell subpopulations (e.g., visceral adipose tissue) and could opens up new diagnostic aspects in the field of obesity. Altogether, the significance of these mostly not yet described genetic aberrations in different fat tissues needs to confirmed in a larger series.

SNP array genomic analysis of matched pairs of brain and liver metastases in primary colorectal cancer.

PURPOSE: Brain metastasis formation is a rare and late event in colorectal cancer (CRC) patients and associated with poor survival. In contrast to other metastatic sites, the knowledge on chromosomal aberrations in brain metastases is very limited. METHODS: Therefore, we carried out single nucleotide polymorphism (SNP) array analyses on matched primary CRC and brain metastases of four patients as well as on liver metastases of three patients. RESULTS: Brain metastases showed more chromosomal aberrations than primary tumors or liver metastases. Commonly occurring aberrations were gain of 8q11.1-q24.3 (primary CRC), gain of 13q12.13-q12.3 (liver metastases), and gain of 20q11.1-q13.33 (brain metastases). Furthermore, we found one copy-neutral loss of heterozygosity (cn-LOH) region on chromosome 3 in primary CRC, three cn-LOH regions in liver metastases and 23 cn-LOH regions in brain metastases, comprising 26 previously undescribed sites. CONCLUSION: The more frequent occurrence of cn-LOHs and subsequently affected genes in brain metastases shed light on the pathophysiology of brain metastasis formation. Further pairwise genetic analyses between primary tumors and their metastases will help to define the role of affected genes in cn-LOH regions.

Functional properties of equine adipose-derived mesenchymal stromal cells cultured with equine platelet lysate.

Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.

Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same.

Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-ß1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Bortezomib sensitizes primary human esthesioneuroblastoma cells to TRAIL-induced apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL), a promising novel anti-cancer cytokine of the TNF superfamily, and Bortezomib, the first-in-class clinically used proteasome inhibitor, alone or in combination have been shown to efficiently kill numerous tumor cell lines. However, data concerning primary human tumor cells are very rare. Using primary esthesioneuroblastoma cells we analyzed the anti-tumor potential and the mechanism employed by Bortezomib in combination with TRAIL for the treatment of this rare but aggressive tumor. Expression of components of the TRAIL pathway was analyzed in tumor specimens and isolated primary tumor cells at the protein level. Cells were treated with TRAIL, Bortezomib, and a combination thereof, and apoptosis induction was quantified. Clonogenicity assays were performed to elucidate the long-term effect of this treatment. Despite expressing all components of the TRAIL pathway, freshly isolated primary esthesioneuroblastoma cells were completely resistant to TRAIL-induced apoptosis. They could, however, be very efficiently sensitized by subtoxic doses of Bortezomib. The influence of Bortezomib on the TRAIL pathway was analyzed and showed upregulation of TRAIL death receptor expression, enhancement of the TRAIL death-inducing signaling complex (DISC), and downregulation of anti-apoptotic proteins of the TRAIL pathway. Of clinical relevance, TRAIL-resistant primary tumor cells could be repeatedly sensitized by Bortezomib, providing the basis for repeated clinical application schedules. This is the first report on the highly synergistic induction of apoptosis in primary esthesioneuroblastoma cells by Bortezomib and TRAIL. This combination, therefore, represents a promising novel therapeutic option for esthesioneuroblastoma.

Molecular analyses of glioblastoma stem-like cells and glioblastoma tissue.

Glioblastoma is a common, malignant brain tumor whose disease incidence increases with age. Glioblastoma stem-like cells (GSCs) are thought to contribute to cancer therapy resistance and to be responsible for tumor initiation, maintenance, and recurrence. This study utilizes both SNP array and gene expression profiling to better understand GSCs and their relation to malignant disease. Peripheral blood and primary glioblastoma tumor tissue were obtained from patients, the latter of which was used to generate GSCs as well as a CD133pos./CD15pos. subpopulation. The stem cell features of GSCs were confirmed via the immunofluorescent expression of Nestin, SOX2, and CD133. Both tumor tissue and the isolated primary cells shared unique abnormal genomic characteristics, including a gain of chromosome 7 as well as either a partial or complete loss of chromosome 10. Individual genomic differences were also observed, including the loss of chromosome 4 and segmental uniparental disomy of 9p24.3→p21.3 in GSCs. Gene expression profiling revealed 418 genes upregulated in tumor tissue vs. CD133pos./CD15pos. cells and 44 genes upregulated in CD133pos./CD15pos. cells vs. tumor tissue. Pathway analyses demonstrated that upregulated genes in CD133pos./CD15pos. cells are relevant to cell cycle processes and cancerogenesis. In summary, we detected previously undescribed genomic and gene expression differences when comparing tumor tissue and isolated stem-like subpopulations.

Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells.

Due to their ability to standardize key physiological parameters, stirred suspension bioreactors can potentially scale the production of quality-controlled pluripotent stem cells (PSCs) for cell therapy application. Because of differences in bioreactor expansion efficiency between mouse (m) and human (h) PSCs, we investigated if conversion of hPSCs, from the conventional "primed" pluripotent state towards the "naïve" state prevalent in mPSCs, could be used to enhance hPSC production. Through transcriptomic enrichment of mechano-sensing signaling, the expression of epigenetic regulators, metabolomics, and cell-surface protein marker analyses, we show that the stirred suspension bioreactor environment helps maintain a naïve-like pluripotent state. Our research corroborates that converting hPSCs towards a naïve state enhances hPSC manufacturing and indicates a potentially important role for the stirred suspension bioreactor's mechanical environment in maintaining naïve-like pluripotency.

Central neurocytoma: SNP array analyses, subtel FISH, and review of the literature.

The central neurocytoma (CN) is a rare brain tumor with a frequency of 0.1-0.5% of all brain tumors. According to the World Health Organization classification, it is a benign grade II tumor with good prognosis. However, some CN occur as histologically "atypical" variant, combined with increasing proliferation and poor clinical outcome. Detailed genetic knowledge could be helpful to characterize a potential atypical behavior in CN. Only few publications on genetics of CN exist in the literature. Therefore, we performed cytogenetic analysis of an intraventricular neurocytoma WHO grade II in a 39-year-old male patient by use of genome-wide high-density single nucleotide polymorphism array (SNP array) and subtelomere FISH. Applying these techniques, we could detect known chromosomal aberrations and identified six not previously described chromosomal aberrations, gains of 1p36.33-p36.31, 2q37.1-q37.3, 6q27, 12p13.33-p13.31, 20q13.31-q13.33, and loss of 19p13.3-p12. Our case report contributes to the genetic knowledge about CN and to increased understanding of "typical" and "atypical" variants.

Bortezomib sensitizes primary human astrocytoma cells of WHO grades I to IV for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

PURPOSE: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. EXPERIMENTAL DESIGN: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. RESULTS: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomib-enhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIP(L) and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. CONCLUSION: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.

Concise Review: Molecular Cytogenetics and Quality Control: Clinical Guardians for Pluripotent Stem Cells.

Now that induced pluripotent stem cell (iPSC)-based transplants have been performed in humans and organizations have begun producing clinical-grade iPSCs, it is imperative that strict quality control standards are agreed upon. This is essential as both ESCs and iPSCs have been shown to accumulate genomic aberrations during long-term culturing. These aberrations can include copy number variations, trisomy, amplifications of chromosomal regions, deletions of chromosomal regions, loss of heterozygosity, and epigenetic abnormalities. Moreover, although the differences between iPSCs and ESCs appear largely negligible when a high enough n number is used for comparison, the reprogramming process can generate further aberrations in iPSCs, including copy number variations and deletions in tumor-suppressor genes. If mutations or epigenetic signatures are present in parental cells, these can also be carried over into iPSCs. To maximize patient safety, we recommend a set of standards to be utilized when preparing iPSCs for clinical use. Reprogramming methods that do not involve genomic integration should be used. Cultured cells should be grown using feeder-free and serum-free systems to avoid animal contamination. Karyotyping, whole-genome sequencing, gene expression analyses, and standard sterility tests should all become routine quality control tests. Analysis of mitochondrial DNA integrity, whole-epigenome analyses, as well as single-cell genome sequencing of large cell populations may also prove beneficial. Furthermore, clinical-grade stem cells need to be produced under accepted regulatory good manufacturing process standards. The creation of haplobanks that provide major histocompatibility complex matching is also recommended to improve allogeneic stem cell engraftment. Stem Cells Translational Medicine 2018;7:867-875.

Comprehensive cytogenetic characterization of an esthesioneuroblastoma.

Esthesioneuroblastoma is a malignant neuroectodermal tumor originating from olfactory epithelial cells in the nasal vault. Due to the rarity of this tumor entity, cytogenetic data are very limited. Therefore, we performed comprehensive cytogenetic analyses of an esthesioneuroblastoma, Hyam's grade III-IV, using trypsin-Giemsa staining (GTG banding), multicolor fluorescence in situ hybridization (M-FISH), and locus-specific FISH complemented by molecular karyotyping using high-density single nucleotide polymorphism arrays. GTG banding of 25 metaphases revealed 54 structural intrachromosomal aberrations, predominantly located on 2q, 6q, 21q, and 22q, which were confirmed by FISH analysis. Interestingly, we found two novel, so far not described deletions, del(2)(q37) and del(21)(q22). Using GTG banding, locus-specific FISH, and M-FISH, we detected numeric changes of chromosomes 5, 17, 19, and 22, as well as trisomy 8 at low frequency. Applying SNP array karyotyping, we confirmed the chromosomal aberrations del(2)(q37.3), del(3)(q27.2), del(10)(q26.11), chromosomal imbalance on 17q, del(21)(q22), and revealed a number of so far unknown aberrations (gain of 2q14.3, 13q33.3, and 13q34). While the cytogenetically revealed low frequency mosaic del(6)(q22q24) was not visible using SNP array karyotyping, some of the smaller imbalances (SNP array data) could not have been detected by classic cytogenetic analysis. Therefore, our study supports the usefulness of applying complementary methods for cytogenetic analysis.

Cytogenetic and molecular biological characterization of an adult medulloblastoma.

Medulloblastoma is a malignant invasive embryonal tumor, occurring in children mainly. It is rare in adults (<1% of adult brain tumors), and so comprehensive cytogenetic and molecular biological data on adult medulloblastomas are very limited. Conventional therapies provide disappointing long-term disease control, and new therapeutic options are being tested. We performed comprehensive cytogenetic analyses of an adult medulloblastoma, WHO grade IV, using trypsin-Giemsa staining (GTG-banding), multicolor fluorescence in situ hybridization (M-FISH), and locus-specific FISH, complemented by molecular karyotyping using high-density single nucleotide polymorphism (SNP) arrays. GTG-banding of 25 metaphases revealed 31 structural chromosomal aberrations, predominantly located on chromosomes 4q, 9q, 10q, 11p, and 20q, which were confirmed by M-FISH. Two novel, so far not described translocations were found: t(4;11)(q25;p15) and t(9;20)(p23;p12). GTG-banding, locus-specific FISH, and M-FISH detected numerical changes of chromosomes 8, 14, 18, 19, 20, 21, and 22. Molecular karyotyping by SNP array confirmed chromosomal changes -2p, -10q, -16q, and -Xq and revealed de novo partial uniparental disomy 1q and 9q. Applying an upcoming therapeutic approach, we found that primary medulloblastoma cells were resistant to TRAIL, a novel anticancer cytokine, but could be efficiently sensitized by cotreatment with the proteasome inhibitor bortezomib. Bortezomib-TRAIL cotreatment may serve as a powerful therapeutic option for medulloblastoma patients.

Comparative genomic hybridization pattern of non-anaplastic and anaplastic oligodendrogliomas--a meta-analysis.

Many oligodendrogliomas (ODG) have been investigated by comparative genomic hybridization (CGH). To visualize characteristic aberration profiles of non-anaplastic in a comparison with anaplastic ODGs, we performed a meta-analysis of the CGH results of all 89 cases published so far. Therefore, we expanded all given aberrations to the maximum of 850 GTG band resolution. The frequencies of each chromosomal band affected by a genetic imbalance were calculated for WHO grades II and III separately. In non-anaplastic ODGs, -1p and -19q were the most prominent aberrations. In anaplastic ODGs, +7, -4q, -9p, -10, and -15q emerged additionally. We could confirm the existence of three disjunct genetically defined subgroups of ODGs, characterized by -1p/-19q (n=58, 65%, subgroup A), +7/-10 (n=6, 7%, subgroup B) or the absence of either of the two patterns (n=25, 28%, subgroup C). Interestingly, we found a unique aberration pattern in subgroup C (-1p31, -4q, -11p15, -18q, -22q, +17p, +17q) that was different from subgroups A and B, which could indicate a unique molecular carcinogenetic pathway of this ODG subset. Scrutinizing published putative progression markers of ODG, we found that only +7, -10, and -15q significantly correlated with a higher grade of malignancy. Summing up, the expansion of the CGH results to the 850 GTG band resolution enabled a meta-analysis to visualize WHO grade-specific aberration profiles in ODG for the first time.

WHO grade related expression of TRAIL-receptors and apoptosis regulators in meningioma.

BACKGROUND AND AIMS: The expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and key regulators of the extrinsic apoptosis pathway correlate with clinical features and the WHO grade of malignancy in some tumor entities. Expression of pro-apoptotic TRAIL receptors and executioners of apoptosis are a prerequisite for TRAIL-based therapies as a promising future targeted therapy. METHODS: Human meningioma tissues (n=24 WHO grade I, n=7 WHO grade II, n=6 WHO grade III) were immunohistochemically analyzed for the expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, caspase-8, cFLIP, Bcl-2, Bcl-XL, Mcl-1, Bax, and Bak. Staining intensities were quantified by an automated software-based algorithm. RESULTS: While TRAIL-R1 and TRAIL-R3 were nearly absent in meningiomas, TRAIL-R2 and TRAIL-R4 were abundantly expressed. However, only TRAIL-R4 expression correlated with the WHO grade of malignancy. Bcl-2 showed a non-significant upregulation in WHO grade III meningiomas. Bcl-XL and Mcl-1 expression was significantly higher in WHO grade II compared to grade I. Bcl-XL and TRAIL-R4 expression correlated with the mitotic activity (Ki67) of the tumor. Furthermore, TRAIL-R2 expression correlated with TRAIL-R4. Bak expression correlated with both, Bcl-XL and Mcl-1 expression. The expression patterns did neither correlate with the progression-free nor with the overall survival of the meningioma patients. CONCLUSIONS: Apoptosis-inducing TRAIL-R2 and all key executioners of the extrinsic apoptosis pathway are abundantly expressed in meningioma. For some regulators of apoptosis with opposite functions, the expression of the pro-apoptotic protein significantly correlated with the expression level of the respective anti-apoptotic binding partner, possibly resulting in a steady-state of apoptosis. TRAIL-R2 might serve as a novel therapeutic target in meningioma.

Molecular breakpoint analysis and relevance of variable mosaicism in a woman with short stature, primary amenorrhea, unilateral gonadoblastoma, and a 46,X,del(Y)(q11)/45,X karyotype.

We report on a 30-year-old woman with short stature, completely female external genitalia, primary amenorrhea, bilateral streak gonads, unilateral gonadoblastoma, and a 46,X,del(Y)(q11)/45,X karyotype. Variable levels of mosaicism were found in blood and cultivated fibroblasts from both the skin and ovaries, with the percentage of the 45,X lineage never exceeding 33%. Fluorescence in situ hybridization (FISH) was performed with alpha satellite centromere region probes of the X and Y chromosomes (DXZ1 and DXZ3) as well as with the unique-sequence, locus-specific, sex-determining region of the Y chromosome gene (SRY) and the DXZ1 probes. Each signal was noted for DXZ1 on the X chromosome and for the Y probes on the marker chromosome. Molecular investigations with a panel of PCR markers spread over the whole Y chromosome indicated a deletion breakpoint between sY 78 (interval 4) and sY 151 (interval 5F). No mutation of the high mobility group-box (HMG-box) of the SRY gene could be found following sequence analysis. The phenotype/genotype correlation demonstrates the broad phenotypic range of low-level 45,X mosaicism with the resultant short stature and external female phenotype, despite the presence of SRY in a high proportion of cells in various tissues.

In vivo transplantation of neurosphere-like bodies derived from the human postnatal and adult enteric nervous system: a pilot study.

Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via 'bystander' mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.