Local Application of Isogenic Adipose-Derived Stem Cells Restores Bone Healing Capacity in a Type 2 Diabetes Model.

UNLABELLED: Bone regeneration is typically a reliable process without scar formation. The endocrine disease type 2 diabetes prolongs and impairs this healing process. In a previous work, we showed that angiogenesis and osteogenesis-essential steps of bone regeneration-are deteriorated, accompanied by reduced proliferation in type 2 diabetic bone regeneration. The aim of the study was to improve these mechanisms by local application of adipose-derived stem cells (ASCs) and facilitate bone regeneration in impaired diabetic bone regeneration. The availability of ASCs in great numbers and the relative ease of harvest offers unique advantages over other mesenchymal stem cell entities. A previously described unicortical tibial defect model was utilized in diabetic mice (Lepr(db-/-)). Isogenic mouse adipose-derived stem cells (mASCs)(db-/db-) were harvested, transfected with a green fluorescent protein vector, and isografted into tibial defects (150,000 living cells per defect). Alternatively, control groups were treated with Dulbecco's modified Eagle's medium or mASCs(WT). In addition, wild-type mice were identically treated. By means of immunohistochemistry, proteins specific for angiogenesis, cell proliferation, cell differentiation, and bone formation were analyzed at early (3 days) and late (7 days) stages of bone regeneration. Additionally, histomorphometry was performed to examine bone formation rate and remodeling. Histomorphometry revealed significantly increased bone formation in mASC(db-/db-)-treated diabetic mice as compared with the respective control groups. Furthermore, locally applied mASCs(db-/db-) significantly enhanced neovascularization and osteogenic differentiation. Moreover, bone remodeling was upregulated in stem cell treatment groups. Local application of mACSs can restore impaired diabetic bone regeneration and may represent a therapeutic option for the future. SIGNIFICANCE: This study showed that stem cells obtained from fat pads of type 2 diabetic mice are capable of reconstituting impaired bone regeneration in type 2 diabetes. These multipotent stem cells promote both angiogenesis and osteogenesis in type 2 diabetic bony defects. These data might prove to have great clinical implications for bony defects in the ever-increasing type 2 diabetic patient population.

Surgical Debridement Is Superior to Sole Antibiotic Therapy in a Novel Murine Posttraumatic Osteomyelitis Model.

INTRODUCTION: Bone infections after trauma, i.e. posttraumatic osteomyelitis, pose one of the biggest problems of orthopedic surgery. Even after sufficient clinical therapy including vast debridement of infected bone and antibiotic treatment, regeneration of postinfectious bone seems to be restricted. One explanation includes the large sized defects resulting from sufficient debridement. Furthermore, it remains unclear if inflammatory processes after bone infection do affect bone regeneration. For continuing studies in this field, an animal model is needed where bone regeneration after sufficient treatment can be studied in detail. METHODS: For this purpose we created a stable infection in murine tibiae by Staphylococcus aureus inoculation. Thereafter, osteomyelitic bones were debrided thoroughly and animals were subsequently treated with antibiotics. Controls included debrided, non-infected, as well as infected animals exclusively treated with antibiotics. To verify sufficient treatment of infected bone, different assessments detecting S. aureus were utilized: agar plates, histology and RT-qPCR. RESULTS: All three detection methods revealed massive reduction or eradication of S. aureus within debrided bones 1 and 2 weeks postoperatively, whereas sole antibiotic therapy could not provide sufficient treatment of osteomyelitic bones. Debrided, previously infected bones showed significantly decreased bone formation, compared to debrided, non-infected controls. DISCUSSION: Thus, the animal model presented herein provides a reliable and fascinating tool to study posttraumatic osteomyelitis for clinical therapies.

Quantitative RT-PCR specific for precursor and mature miRNAs.

Quantification of microRNAs (miRNAs) in cells or primary tissues is one of the most important steps in elucidating their biological functions. However, miRNAs are challenging molecules for PCR amplification due to the stable hairpin of the precursor form and the small size of the mature miRNA, which is roughly the same length as the primers used in standard PCR. To date, different assays were introduced for the specific and sensitive quantification of the mature form of miRNAs. In this chapter we describe the extraction of RNA from microdissected tissue and the quantification of miRNAs using two different methods (stem-loop qRT-PCR and polyT adaptor qRT-PCR).

Lentiviral overexpression of miRNAs.

Deregulation of microRNAs (miRNAs) has been attributed to almost any human disease analyzed to date. This calls for models and experimental strategies for functional analyses of miRNAs enabling miRNA overexpression or suppression in target cell and/or tissues. Lentiviral vector (LV)-based technologies allow the long-term overexpression of miRNAs in nearly all cell types in an easy and rapid manner and are therefore popular tools for this application. In this chapter we describe the cloning of LV miRNA expression vectors as well as the production of virus particles for target cell infection and stable expression of miRNAs.

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma.

Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2-1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2-1f) and not from miR-1246. In addition, we observed a remarkable stability of RNU2-1f in serum and provide experimental evidence that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

MiR-30a-5p suppresses tumor growth in colon carcinoma by targeting DTL.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in different biological processes by suppressing target gene expression. Altered expression of miR-30a-5p has been reported in colon carcinoma. To elucidate its potential biological role in colon cancer, miR-30a-5p was overexpressed via a lentiviral vector system in two different colon cancer cell lines. This induced in both lines miR-30a-5p-mediated growth inhibition, attributable to a cell cycle arrest at the G(1) phase and an induction of apoptosis. Combining global gene expression analyses of miR-30a-5p transgenic line HCT116 with in silico miRNA target prediction, we identified the denticleless protein homolog (DTL) as a potential miRNA-30a-5p target. Subsequent reporter gene assays confirmed the predicted miR-30a-5p binding site in the 3'untranslated region of DTL. Importantly, overexpression of DTL in HCT116 cells partially rescued these cells from miR-30a-5p-mediated growth suppression. In addition, TP53 and CDKN1A expression were increased in miR-30a-5p-overexpressing HCT116 cells, suggesting that miR-30a-5p is able to modulate the cell cycle via a DTL-TP53-CDKN1A regulatory circuit. Finally, 379 colorectal cancer tissues were screened for DTL expression and DTL was found to be overexpressed in 95.8% of human colorectal cancers compared with normal colon mucosa. In conclusion, our data identified miR-30a-5p as a tumor-suppressing miRNA in colon cancer cells exerting its function via modulation of DTL expression, which is frequently overexpressed in colorectal cancer. Thus, our data suggest that restoring miR-30a-5p function may prove useful as therapeutic strategy for tumors with reduced miR-30a-5p expression.

Identification of microRNAs in the cerebrospinal fluid as biomarker for the diagnosis of glioma.

Malignant gliomas are the most common and lethal primary intracranial tumors. To date, no reliable biomarkers for the detection and risk stratification of gliomas have been identified. Recently, we demonstrated significant levels of microRNAs (miRNAs) to be present in cerebrospinal fluid (CSF) samples from patients with primary CNS lymphoma. Because of the involvement of miRNA in carcinogenesis, miRNAs in CSF may serve as unique biomarkers for minimally invasive diagnosis of glioma. The objective of this pilot study was to identify differentially expressed microRNAs in CSF samples from patients with glioma as potential novel glioma biomarkers. With use of a candidate approach of miRNA quantification by reverse-transcriptase polymerase chain reaction (qRT-PCR), miRNAs with significant levels in CSF samples from patients with gliomas were identified. MiR-15b and miR-21 were differentially expressed in CSF samples from patients with gliomas, compared to control subjects with various neurologic disorders, including patients with primary CNS lymphoma and carcinomatous brain metastases. Receiver-operating characteristic analysis of miR-15b level revealed an area under the curve of 0.96 in discriminating patients with glioma from patients without glioma. Moreover, inclusion of miR-15b and miR-21 in combined expression analyses resulted in an increased diagnostic accuracy with 90% sensitivity and 100% specificity to distinguish patients with glioma from control subjects and patients with primary CNS lymphoma. In conclusion, the results of this pilot study demonstrate that miR-15b and miR-21 are markers for gliomas, which can be assessed in the CSF by means of qRT-PCR. Accordingly, miRNAs in the CSF have the potential to serve as novel biomarkers for the detection of gliomas.

Global microRNA expression profiling of microdissected tissues identifies miR-135b as a novel biomarker for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known for its poor prognosis resulting from being diagnosed at an advanced stage. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs (miRNAs), considered a new class of biomarkers and therapeutic targets, may be able to fulfill those needs. Combining tissue microdissection with global miRNA array analyses, cell type-specific miRNA expression profiles were generated for normal pancreatic ductal cells, acinar cells, PDAC cells derived from xenografts and also from macrodissected chronic pancreatitis (CP) tissues. We identified 78 miRNAs differentially expressed between ND and PDAC cells providing new insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development. Having filtered miRNAs which are upregulated in the three pairwise comparisons of PDAC vs. ND, PDAC vs. AZ and PDAC vs. CP, we identified 15 miRNA biomarker candidates including miR-135b. Using relative qRT-PCR to measure miR-135b normalized to miR-24 in 75 FFPE specimens (42 PDAC and 33 CP) covering a broad range of tumor content, we discriminated CP from PDAC with a sensitivity and specificity of 92.9% [95% CI=(80.5, 98.5)] and 93.4% [95% CI=(79.8, 99.3)], respectively. Furthermore, the area under the curve (AUC) value reached of 0.97 was accompanied by positive and negative predictive values of 95% and 91%, respectively. In conclusion, we report pancreatic cell-specific global miRNA profiles, which offer new candidate miRNAs to be exploited for functional studies in PDAC. Furthermore, we provide evidence that miRNAs are well-suited analytes for development of sensitive and specific aid-in-diagnosis tests for PDAC.

Identification of microRNAs in the cerebrospinal fluid as marker for primary diffuse large B-cell lymphoma of the central nervous system.

The diagnosis of primary central nervous system lymphoma (PCNSL) depends on histopathology of brain biopsies, because disease markers in the cerebrospinal fluid (CSF) with sufficient diagnostic accuracy are not available yet. MicroRNAs (miRNAs) are regulatory RNA molecules that are deregulated in many disease types, including cancer. Recently, miRNAs have shown promise as markers for cancer diagnosis. In this study, we demonstrate that miRNAs are present in the CSF of patients with PCNSL. With a candidate approach and miRNA quantification by reverse transcription polymerase chain reaction, miRNAs with significant levels in the CSF of patients with PCNSL were identified. MiR-21, miR-19, and miR-92a levels in CSF collected from patients with PCNSL and from controls with inflammatory CNS disorders and other neurologic disorders indicated a significant diagnostic value of this method. Receiver-operating characteristic analyses showed area under the curves of 0.94, 0.98, and 0.97, respectively, for miR-21, miR-19, and miR-92a CSF levels in discriminating PCNSL from controls. More importantly, combined miRNA analyses resulted in an increased diagnostic accuracy with 95.7% sensitivity and 96.7% specificity. We also demonstrated a remarkable stability of miRNAs in the CSF. In conclusion, CSF miRNAs are potentially useful tools as novel noninvasive biomarker for the diagnosis of PCNSL.