Alu Gene Research Articles

Using qPCR and ddPCR to study biodistribution of cell therapy products: a multi-site evaluation

Backgroud aimsRegenerative therapies employing cell therapy products (CTPs) have attracted considerable attention. Biodistribution (BD) evaluation of CTPs is mainly performed to clarify the cell survival time, engraftment, and distribution site. This evaluation is crucial for predicting the efficacy and safety profiles of clinical studies based on non-clinical BD study outcomes. However, no internationally unified method has been established for assessing cell BD after administration. Here, we aimed to standardize the BD assay method used for CTPs, conducting the following evaluations using the same protocol across multiple study facilities: (1) in vitro validation of quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) analyses using the primate-specific Alu gene, and (2) in vivo BD studies after the intravenous administration of human mesenchymal stem cells (hMSCs) to immunodeficient mice, commonly used in non-clinical tumorigenicity studies. MethodsQuality control samples were prepared and analyzed by adding a fixed number of human-derived cells to several mouse tissues. The respective quantitative performances of the qPCR and ddPCR methods were compared for accuracy and precision. hMSCs were intravenously administered to immunodeficient mice, and tissues were collected at 1, 4, and 24 h after administration. ResultsBoth methods demonstrated an accuracy (relative error) generally within ±50% and a precision (coefficient of variation) generally less than 50%. While differences in calibration curve ranges were observed between qPCR and ddPCR, no significant differences in quantification were found among the assay facilities. The BD of hMSCs in mice was evaluated at seven facilities (qPCR at three facilities; ddPCR at four facilities), revealing similar tissue distribution profiles in all facilities, with the lungs showing the highest cell distribution among the tissues tested. ConclusionsQuantitative evaluation of qPCR and ddPCR using Alu sequences was conducted, demonstrating that the test method can be adapted for BD evaluation.

Combination of Circulating Cell-Free DNA and Positron Emission Tomography to Distinguish Non-Small Cell Lung Cancer from Tuberculosis.

Non-small cell lung cancer (NSCLC) holds high metabolic tumor burden and circulating cell-free DNA (cfDNA) levels, and the relationship between metabolic tumor burden and cfDNA in NSCLC and the underlying mechanism of their interaction therein remain poorly characterized. Our aim was to evaluate the clinical value of cfDNA and metabolic tumor burden by positron emission tomography-computed tomography (PET/CT) for NSCLC differential diagnosis from tuberculosis in patients with solitary pulmonary nodules. Metabolic tumor burden values in humans (subjects with NSCLC, subjects with tuberculosis, and healthy control subjects) and relevant mouse models were detected by preoperative 18F-fluorodeoxyglucose PET (18F-FDG PET/CT) and [3H]-2-deoxy-DG uptake, respectively. The cfDNA levels were detected by quantifying serum cfDNA fragments from the ALU (115 bp) gene using reverse transcription-polymerase chain reaction. RNA sequence was performed to determine the underlying target genes and knocked down or inhibited the target genes in vivo and in vitro to determine the mechanism therein. Metabolic tumor burden correlated with serum cfDNA levels in NSCLC subjects but not in tuberculosis subjects or healthy controls. Mouse models showed a similar phenomenon. In addition, the RNA sequence showed that glucose transporter 1 (GLU1), factor-related apoptosis ligand (FasL), caspase 8, and caspase 3 were significantly increased in NSCLC mouse tumors compared with those in tuberculosis mouse masses. Inhibiting the metabolic tumor burden by blocking or knocking down GLU1 markedly reduced the expression of FasL, the phosphorylation of caspase 8/caspase 3, and serum cfDNA levels/apoptosis percentage in vivo and in vitro. Furthermore, the use of a combination of cfDNA and metabolic tumor burden allowed better ability to distinguish NSCLC subjects from those with tuberculosis or healthy controls than either method used alone. Metabolic tumor burden promotes the formation of circulating cfDNA through GLU1-mediated apoptosis in NSCLC, and the combination of cfDNA and metabolic tumor burden could be valuable for distinguishing NSCLC from tuberculosis.

The integrity of cfDNA in follicular fluid and spent medium from embryo culture is associated with embryo grade in patients undergoing in vitro fertilization.

This study was conducted to verify if the cfDNA integrity (cfDI) in follicular fluid and subsequent spent embryo medium (SEM) could serve as potential non-invasive biomarker for high-grade embryo selection during IVF/ICSI. Thirty-two follicular fluids, 32 subsequent corresponding cleavage embryo SEM, and 23 subsequent blastocyst SEM were collected from 11 patients undergoing IVF/ICSI. CfDI was measured by ALU gene amplicons with different sizes by qPCR, as the ratio of long to short fragments. CfDI in follicular fluid corresponding to subsequent high-grade cleavage embryos and blastocysts was significantly lower than that related to low-grade embryos (p = 0.018). Conversely, cfDI in SEM was significantly and positively correlated with high-grade embryos at both stages (p = 0.009). ROC curves of the analysis of cfDI in follicular fluid showed great potential in predicting subsequent embryogenesis and embryo grade (AUC > 0.927). Regardless of the cleavage embryo grade by morphology, cfDI in day 3 SEM could predict if the cleavage embryo could develop to a high-grade blastocyst (AUC = 0.820). A concordant shift pattern of cfDI from follicular fluid to subsequent day 3 SEM and day 5 SEM was found in 81.82% participants featured by various clinical characteristics. CfDI in follicular fluid and SEM was significantly correlated with embryogenesis and embryo grade and could serve as a potential non-invasive biomarker in high-grade embryo selection. Direct qPCR was proved as a labor-saving and sensitive method for the analysis of cfDI in low volume of SEM.

Association of plasma DNA integrity and long fragment ALU247 in the diagnosis of epithelial ovarian cancer

DNA Integrity index (DNA Int) and cell-free DNA (cf-DNA) represent promising biomarkers for epithelial ovarian cancer (EOC) detection. Tumor necrosis produces DNA fractions of different sizes, which contrasts apoptosis in normal tissue that releases smaller and more regular DNA fragments. Using ALU gene primers in quantitative PCR, the amplified cf-DNA is supposed to be either short fragments of 115 bp (ALU 115) or long fragments of 247 bp (ALU 247). ALU levels and DNA Int were determined in the plasma of 30 EOC patients, 30 benign cysts, and 15 healthy individuals. The mean values of DNA Int, ALU115, and ALU247 were elevated in malignant patients (0.51±0.09, 3.93 ng/ul ±1.93, 2.35 ng/ul ±1.1) respectively in comparison to healthy females (0.37±0.05; p < 0.001, 2.56 ng/ul ±0.9; p=0.027, 1.26±0.44; p< 0.01). A significant increase was shown in the mean values of DNA Int and ALU247 of EOC patients compared to those with benign cysts (0.4±0.06, p <0.001; 1.69±0.66, p =0.008) respectively. The area under the curve (AUC) for EOC versus healthy females achieved 0.913 (DNA Int), 0.696 (ALU115), and 0.809 (ALU247) with sensitivities and specificities were (86.7% and 93.3%) for DNA Int, (63.3% and 86.7%) for ALU115 and (76.7% and 86.7%) for ALU247 respectively. Furthermore, comparing patients with EOC versus those with benign cysts gave AUC of 0.834 (DNA Int), 0.564 (ALU115), and 0.681 (ALU247) with sensitivities and specificities were (80% and 80%) for DNA Int, (63.3% and 60%) for ALU115 and (60% and 80%) for ALU247 respectively. Higher DNA Int and plasma ALU247 could help in the assessment of EOC, and their measurements seem to have clinical value in diagnosis.

Gene Conversion amongst Alu SINE Elements.

The process of non-allelic gene conversion acts on homologous sequences during recombination, replacing parts of one with the other to make them uniform. Such concerted evolution is best described as paralogous ribosomal RNA gene unification that serves to preserve the essential house-keeping functions of the converted genes. Transposed elements (TE), especially Alu short interspersed elements (SINE) that have more than a million copies in primate genomes, are a significant source of homologous units and a verified target of gene conversion. The consequences of such a recombination-based process are diverse, including multiplications of functional TE internal binding domains and, for evolutionists, confusing divergent annotations of orthologous transposable elements in related species. We systematically extracted and compared 68,097 Alu insertions in various primates looking for potential events of TE gene conversion and discovered 98 clear cases of Alu–Alu gene conversion, including 64 cases for which the direction of conversion was identified (e.g., AluS conversion to AluY). Gene conversion also does not necessarily affect the entire homologous sequence, and we detected 69 cases of partial gene conversion that resulted in virtual hybrids of two elements. Phylogenetic screening of gene-converted Alus revealed three clear hotspots of the process in the ancestors of Catarrhini, Hominoidea, and gibbons. In general, our systematic screening of orthologous primate loci for gene-converted TEs provides a new strategy and view of a post-integrative process that changes the identities of such elements.

Biodistribution of Intramuscularly-Transplanted Human Dental Pulp Stem Cells in Immunocompetent Healthy Rats through NIR Imaging

Owing to their neural crest origin, dental pulp stem cells (DPSCs) are increasingly gaining prominence in treating nervous system disease conditions. However, as per the regulatory bodies [European-Medicines Agency (EMA), Indian-Council of Medical-Research (ICMR)], their biodistribution after transplantation needs to be evaluated for them to be considered for cell-based therapy for clinical trials. There are yet no studies describing the dynamic distribution of human origin DPSCs (hDPSCs) after transplantation in an immunocompetent, physiologically healthy animal model. Here, using near-infrared (NIR)-based whole animal and ex vivo tissue imaging, we assessed the biodistribution of intramuscularly transplanted hDPSCs in immunocompetent healthy Wistar rats. Further validation was done by quantifying gene expression of the human Alu gene in rat tissues. After 24 h of transplantation, an increase in signal intensity and area of signal was observed in the muscle of administration compared to 30 min and 6 h. At hour 24, neither increase in human Alu nor human Ki67 gene expression was seen in the rat muscle, thus confirming that the increase in signal area and intensity at hour 24 was not due to proliferation of the transplanted cells. Rather at hour 24, the NIR-signal intensity in bone marrow increased, suggesting that the NIR-tagged DPSCs have started entering into the blood vessels adjacent to the muscle, and the blood vessels being placed just beneath the subcutaneous layer might be responsible for an increase in signal intensity. Signal intensity increased distinctly in all organs at this timepoint, confirming that the cells entered the bloodstream by hour 24. Lung entrapment of DPSCs was not observed, since signal intensity was least in lungs as compared to the site of injection. Cells were retained for up to 28 days at the site of injection. These findings lay the basis to design the dosage for intramuscular delivery of hDPSCs for degenerative disease models and for future clinical trials.

Development of a bioanalytical method for circulating human T cells in animals using Arthrobacter luteus-based quantitative polymerase chain reaction and its application in preclinical biodistribution studies.

IntroductionIn the development of cell therapy products for human use, studies on the biodistribution of transplanted cells in animals are important for assessing the safety and efficacy of these products. Although a few reports have described the biodistribution of human cells in animals using Arthrobacter luteus-based-polymerase chain reaction (Alu-PCR), most have used genomic DNA or synthetic oligonucleotide as calibrators, as opposed to actual cells. In addition, bioanalytical variability in the quantification of cells with respect to specificity, selectivity, accuracy, and precision, has not been evaluated. Accordingly, in this study, we validated the utility of this bioanalytical method for human T cells in mice to establish assay performance using cells as a calibrator.MethodsA standard curve was constructed for the addition of cell lysates to mouse tissues and blood, and DNA was extracted. Alu-PCR was applied for the quantification of human peripheral blood CD8+ T cells in mice. To determine assay performance, we evaluated accuracy, precision, selectivity, specificity, and stability. In vivo cell kinetics and biodistribution were investigated based on intravenous administration of human T cells to mice.ResultsAlu-PCR enabled us to specifically detect human T cells in mouse blood and tissues. The lower detection limit of Alu-PCR was 10 cells/15 mg tissue (7.5 mg for spleen and lung) or cells/50 μL blood. Given that PCR threshold cycle (Cq) values among mouse samples (blood, liver spleen, lung, heart, and kidney) show slight variation, calibration curves should be generated using the same tissue as used for the assay. Most coefficients of variation in the assay were within 30%. The cell kinetics of administered human T cells in mice were successfully evaluated using the established Alu-qPCR.ConclusionsThe Alu-PCR technique developed in this study showed sufficient specificity and sensitivity in detecting human peripheral blood CD8+ T cells in mice. This technique, which targets the primate-specific Alu gene, is applicable for quantifying transplanted human cells in animals without the necessity of cell labeling. The data presented herein will be useful for standardizing bioanalytical approaches in biodistribution studies of cell therapy products.

Highly specific, quantitative polymerase chain reaction probe for the quantification of human cells in cynomolgus monkeys

Quantification of human cells may be performed using quantitative polymerase chain reaction (qPCR). In preclinical studies, the human Alu sequence is widely used as biomarker for human DNA. However, because the Alu gene is shared by primates, its use is limited to non-primate studies. The biodistribution of human cells in primates is also necessary for translational studies. Therefore, we aimed to design a novel, human-specific primer/probe that enables the quantification of human cells in primates and other animal models. A novel primer/probe set was successfully designed based on highly repetitive LINE1 sequences. qPCR efficiency (94.95–99.21%) and linearity of calibration curves (r2 = 0.996–0.999) were confirmed in tissue homogenates of cynomolgus monkey. The lower limit of detection was 10 cells per 15-mg tissue sample, a sensitivity that is equivalent to existing Alu primers/probes. The set was also effective in other animal models such as mice, rabbits, pigs, and common marmosets.To our knowledge, this is the first study describing the successful design of a human-specific qPCR primer/probe for human cell quantification in various animals, including non-human primates, using LINE1 sequence. The excellent selectivity, sensitivity, and versatility of the LINE1 primers/probes make it a promising quantification tool in preclinical biodistribution studies.

Directly induced hepatogenic cells derived from human fibroblast ameliorate liver fibrosis.

Recently, reprogramming technology has emerged as a fascinating tool to generate specific tissue cells. In this study, we tested the hypothesis that ultrasound-directed cellular reprogramming can generate fibroblasts into hepatogenic cells. We directly induced human dermal fibroblasts (HDFs) into hepatocyte-like cells mediated by environmental transition-guided cellular reprogramming (h/entr) using ultrasound. We confirmed the characteristics of h/entr by the expression levels of hepatocyte specific RNA and proteins. The effects of h/entr on the activation of hepatic stellate cells were analyzed using conditioned medium (CM). h/entr were transplanted into mice with acute liver fibrosis and the therapeutic effects and mechanism of liver fibrosis were determined. h/entr exhibited high levels of hepatocyte specific genes, hepatogenic (hepatocyte growth factor [HGF], colony-stimulating factor 3 [CSF-3]) and anti-inflammatory (interleukin 10 [IL-10]) factors. h/entr CM suppressed the activation of hepatic stellate cells in vitro. Transplantation of h/entr significantly delayed liver fibrosis and improved liver function. Transplantation of h/entr accelerates liver regeneration, and human albumin expressing h/entr and human Alu gene were detected in the mouse livers. This report suggests that directly induced h/entr could be one of the highly effective therapeutic options for the treatment of liver cirrhotic disease.

Integrity of plasma DNA is inversely correlated with vaccine-induced antitumor immunity in ovarian cancer patients

Cancer immunotherapy including vaccine therapy is a promising modality for cancer treatment, but few patients show its clinical benefits currently. The identification of biomarkers that can identify patients who will benefit from cancer immunotherapy is thus important. Here, we investigated the potential utility of the circulating cell-free DNA (cfDNA) integrity—a ratio of necrotic cell-derived, longer DNA fragments versus apoptotic cell-derived shorter fragments of Alu gene—as a biomarker of vaccine therapy for patients with ovarian cancer. We analyzed plasma samples from 39 patients with advanced or recurrent ovarian cancer enrolled in clinical trials for personalized peptide vaccinations. We observed that (1) the cfDNA integrity was decreased after the first cycle of vaccination, and (2) the decreased levels of cfDNA integrity were correlated with vaccine-induced immune responses; i.e., decreased cfDNA integrity was observed in 91.7% and 59.3% of the IgG-positive and negative patients, respectively (p = 0.0445). Similarly, decreased cfDNA integrity was observed in 92.9% and 56.0% of CTL response-positive and negative patients, respectively (p = 0.0283). These results suggest that the circulating cfDNA integrity is a possible biomarker for cancer vaccine therapy.

Evaluation of cell free circulating plasma DNA in prostate cancer

Background: Prostate cancer (PC) is the most common cancer affecting men, it accounts for 29% of all male cancer and 11% of all male cancer related deaths. DNA is normally released from an apoptotic source which generates small fragments of cell-free DNA, whereas cancer patients have cell-free circulating DNA that originated from necrosis, autophagy, or mitotic catastrophe, which produce large fragments. Aim of work: Differentiate the cell free DNA levels (CFDNA) and its integrity in prostate cancer patients and control group composed of benign prostate hyperplasia (BPH) and healthy persons. Methodology: cf-DNA levels were quantified by real-time PCR amplification in prostate cancer patients (n = 50), (BPH) benign prostate hyperplasia (n = 25) and healthy controls (n = 30) using two sets of ALU gene (product size of 115 bp and 247-bp) and its integrity was calculated as a ratio of qPCR results of 247 bp ALU over 115 bp ALU. Results: Highly significant levels of cf-DNA and its integrity in PC patients compared to BPH. Twenty-eight (56%) patients with prostate cancer had bone metastasis. ALU115 q PCR is superior to the other markers in discriminating metastatic patients with a sensitivity of 96.4% and a specificity of 86.4% (AUC = 0.981) Conclusion: ALU115 q PCR could be used as a valuable biomarker helping in identifying high risk patients, indicating early spread of tumor cells as a potential seed for future metastases.

Abstract 1472: Alternate translation initiation regulation of PRDX5 mRNA by miR6855<->3p in basal<->like breast cancer cells

Abstract Human peroxiredoxins (PRDXs) are a superfamily of six thiol<->dependent peroxidases that are able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. Apart from the classical, apparently redundant function of mitochondrial and often peroxisomal peroxide neutralization shared by other peroxiredoxins, PRDX5 was initially discovered as a DNA binding transcriptional repressor regulating the biosynthesis of cytotoxic Alu RNAs through a mitochondrial<->localization signal<->truncated mature form (SPRDX5] of this protein that is accumulated in the nucleus and binds to a 60 bp nucleotide sequence at the Alu gene promoters. Our data suggest that SPRDX5 also binds to the Alu<->sequence<->containing BRCA2 gene silencer to prevent the binding of SLUG at the silencer thus enhancing the BRCA2 gene expression leading to the genotoxin and radiation<->resistance of aggressive SLUG<->high basal<->like breast cancer cells. PRDX5 mRNA contains two in<->frame start codons (AUGs) that are conditionally used as alternate translation initiation sites. Translation from the first AUG would result in the synthesis of a larger 214<->residue protein (LPRDX5) whereas the use of the second AUG would result in the production of a shorter 162<->residue polypeptide (SPRDX5). Since SPRDX5 lacks the mitochondrial localization signal, it is accumulated in the nucleus via its C<->terminal bipartite nuclear localization signal. We present evidence here that a miRNA, miR<->6855<->3p, binds the primary PRDX5 transcript between the two AUG codons and prevents the translation of the mRNA from the first AUG codon but not that from the second AUG codon thus favoring the initiation of translation from the second AUG codon. To characterize the effect of miR<->6855<->3p on Prdx5 mRNA translation, we developed three different PRDX5<->FLAG constructs in pCV3XFLAG14 (Sigma) that has Prdx5 ORF with or without mutation on the first or second AUG codon. Transfection of these constructs into BT549 cells followed by treatments with miR<->6855<->3p mimic or antagomiR and subcellular fractionation further supported our notion. We propose here that miR<->6855<->3p acts as a tsmiR by promoting the biosynthesis of the transcriptional regulator protein SPRDX5 which enhances the expression of the tumor suppressor protein BRCA2 leading to the genotoxin and radiation<->resistance of aggressive SLUG<->high basal<->like breast cancer cells. Supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920 01 to GC and 1U54RR026140 to SM. Citation Format: Gautam Chaudhuri, Smita Misra. Alternate translation initiation regulation of PRDX5 mRNA by miR6855<->3p in basal<->like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2017-1472

Quantitative analysis of plasma cell-free DNA and its DNA integrity in patients with metastatic prostate cancer using ALU sequence

BackgroundProstate cancer (PC) is the most common cancer affecting men, it accounts for 29% of all male cancer and 11% of all male cancer related death. DNA is normally released from an apoptotic source which generates small fragments of cell-free DNA, whereas cancer patients have cell-free circulating DNA that originated from necrosis, autophagy, or mitotic catastrophe, which produce large fragments. Aim of workDifferentiate the cell free DNA levels (cfDNA) and its integrity in prostate cancer patients and control group composed of benign prostate hyperplasia (BPH) and healthy persons. Methodologycf-DNA levels were quantified by real-time PCR amplification in prostate cancer patients (n = 50), (BPH) benign prostate hyperplasia (n = 25) and healthy controls (n = 30) using two sets of ALU gene (product size of 115bp and 247-bp) and its integrity was calculated as a ratio of qPCR results of 247bp ALU over 115bp ALU. ResultsHighly significant levels of cf-DNA and its integrity in PC patients compared to BPH. Twenty-eight (56%) patients with prostate cancer had bone metastasis. ALU115 qpcr is superior to the other markers in discriminating metastatic patients with a sensitivity of 96.4% and a specificity of 86.4% and (AUC=0.981) ConclusionALU115 qpcr could be used as a valuable biomarker helping in identifying high risk patients, indicating early spread of tumor cells as a potential seed for future metastases.

Establishment of Humanized Mouse Model by Using Transplantation of Mobilized Peripheral Blood Stem Cells

To investigate the hematopoietic reconstitution in immunodeficiency NPG(TM) mice after transplantation of G-CSF-mobilized peripheral blood CD34(+) hemopoietic stem cells. CD34(+) cells were isolated from peripheral blood stem cells (PBSC) by magnetic activated cell sorting (MACS), and then were transplanted into NPG(TM) mice irradiated with sublethal dose of X ray by marrow cavity transplantation. The hemogram of mice after transplantation for 2, 4 weeks was observed; human cell populations (CD45(+), CD19(+)) in the peripheral blood of mice were dynamically analyzed by flow cytometry (FCM) at 4, 6, 8, 10 and 12 weeks after transplantation. Until the planned harvest at the 12 week after transplantation, the CD45(+), CD19(+) level in bone marrow, liver, spleen from each mouse were detected by flow cytometry; the expression of human Alu gene in the bone marrow cell of mouse was detected by PCR. The purity of CD34(+) cells accounted for 96.3%; after irradiation, the nucleated cells and megalokaryocytes in the marrow cavity of NPG mice were reduced significantly or were lost, and reached the myeloablative effect. At week 4 after transplantation, components of blood cells in peripheral blood of transplanted mice were recovered to the level before irradiation; all the mice survived, human CD45(+), CD19(+) cells were found by FCM in the peripheral blood of all the surviving mice in transplantation group at week 4, 6, 8, 10, 12 after the transplantation; at the 12th week, the human Alu gene could be detected in the bone marrow of all the mice in transplantation group. The human-mouse chimeric model is successfully established in irradiation-induced NPG mouse by transplantation of CD34(+) HSC from G-CSF-mobilized peripheral blood via marrow cavity.

ROLE OF ADIPOSE DERIVED MESENCHYMAL STEM CELLS IN IMPROVING STREPTOZOTOCIN-INDUCED DIABETES MELLITUS IN RATS

Background: Diabetes mellitus (DM) is one of the most recognizable endocrine metabolic disorders characterized by chronic hyperglycemia.Mesenchymalstem cells (MSCs) are promising toolto treatDM type I (DM 1). They can be isolated from many sources including adipose tissue because of its easy availability. Objective: Evaluation of the effect of adipose derived MSCs (ADMSCs) on streptozotocin (STZ)-induced DM in rats. Materials and Methods: Forty eight adult male albino rats were included and divided into 4 equal groups. Group I: control untreated, group II: diabetic group, group III: insulin-treated group, and group IV: ADMSCs-treated group. After 21 days from treatment, animals were sacrificed and pancreas was obtained for histological assessments. Stem cell homing was detected by polymerase chain reaction for human specific Alu gene. Results: ADMSCs caused lowering of blood glucose levels starting at the 14th day of treatment and increased blood insulin levels accompanied by reversal of the histopathological changes that were found in the diabetic group and increase insulin secreting cells. Conclusion: ADMSCs improved blood glucose levels and reversed the histopathological changes of the pancreas in experimentally induced DM I.

Validation of a Quantitative Polymerase Chain Reaction Method for Human Alu Gene Detection in Microchimeric Pigs Used as Donors for Xenotransplantation

This work was undertaken to evaluate whether a real-time quantitative polymerase chain reaction (qPCR) is as an adequate method for detection and quantification of human-specific DNA elements (Alu gene) in tissues and blood samples of pigs in which human stem cells were engrafted. Real-time qPCR quantification was performed with the use of previously described primers. The human DNA was mixed with different quantities of porcine DNA. The primer concentration and specificity, the qPCR efficiency, the quantification variations due to different porcine DNA concentrations, and the dissociation curve produced by the assay were evaluated. The qPCR proved to be specific, robust, with a reproducible and specific bimodal melting curve. High porcine DNA concentration produced subquantification, especially with low human DNA quantity. However, the assay proved to be useful for the detection of chimeric piglets produced by human cells injected in utero, because the effect caused by the porcine DNA interference was corrected in quantification of human DNA from piglets.

New adipose tissue formation by human adipose-derived stem cells with hyaluronic acid gel in immunodeficient mice.

Background: Currently available injectable fillers have demonstrated limited durability. This report proposes the in vitro culture of human adipose-derived stem cells (hASCs) on hyaluronic acid (HA) gel for in vivo growth of de novo adipose tissue.Methods: For in vitro studies, hASCs were isolated from human adipose tissue and were confirmed by multi-lineage differentiation and flow cytometry. hASCs were cultured on HA gel. The effectiveness of cell attachment and proliferation on HA gel was surveyed by inverted light microscopy. For in vivo studies, HA gel containing hASCs, hASCs without HA gel, HA gel alone were allocated and subcutaneously injected into the subcutaneous pocket in the back of nude mice (n=6) in each group. At eight weeks post-injection, the implants were harvested for histological examination by hematoxylin and eosin (H&E) stain, Oil-Red O stain and immunohistochemical staining. The human-specific Alu gene was examined.Results: hASCs were well attachment and proliferation on the HA gel. In vivo grafts showed well-organized new adipose tissue on the HA gel by histologic examination and Oil-Red O stain. Analysis of neo-adipose tissues by PCR revealed the presence of the Alu gene. This study demonstrated not only the successful culture of hASCs on HA gel, but also their full proliferation and differentiation into adipose tissue.Conclusions: The efficacy of injected filler could be permanent since the reduction of the volume of the HA gel after bioabsorption could be replaced by new adipose tissue generated by hASCs. This is a promising approach for developing long lasting soft tissue filler.

Repeated Systemic Administration of Human Adipose-Derived Stem Cells Attenuates Overt Diabetic Nephropathy in Rats

Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5×10(6) human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.

Plasma cell-free DNA and its DNA integrity as biomarker to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate-specific antigen

To investigate whether plasma cell-free DNA (cfDNA) or its integrity could differentiate prostate cancer from benign prostate hyperplasia (BPH) in patients with serum prostate-specific antigen (PSA) ≥ 4 ng/ml. Ninety-six patients with prostate cancer and 112 patients with BPH were enrolled. cfDNA levels in plasma before prostate biopsy were quantified by real-time PCR amplification of ALU gene (product size of 115 bp), and quantitative ratio of ALU (247 bp) to ALU (115 bp) reflected the integrity of cfDNA. In patients with serum PSA ≥ 4 ng/ml, there were significant differences in plasma cfDNA or its integrity between the patients with prostate cancer (19.74 ± 4.43, 0.34 ± 0.05) and patients with BPH (7.36 ± 1.58, 0.19 ± 0.03; P < 0.001, P < 0.001). Prostate cancer could be differentiated with a sensitivity of 73.2 % and a specificity of 72.7 % by cfDNA (AUC = 0.864). The integrity of cfDNA had a sensitivity of 81.7 % and a specificity of 78.8 % for the distinguishing prostate cancer from BPH (AUC = 0.910). cfDNA and its integrity could be applied to differentiate prostate cancer from BPH in patients with serum PSA ≥ 4 ng/ml.

Human Mesenchymal Stromal Cells: Identifying Assays to Predict Potency for Therapeutic Selection

Multipotent mesenchymal stromal cells (MSCs) have the potential to repair and regenerate damaged tissues, making them attractive candidates for cell-based therapies. To maximize efficacy of MSCs, prediction of their therapeutic abilities must be made so that only the best cells will be used. Our goal was to identify feasible and reproducible in vitro assays to predict MSC potency. We generated cell lines from 10 normal human bone marrow samples and used the International Society for Cellular Therapy's minimal criteria to define them as MSCs: plastic adherence, appropriate surface marker expression, and trilineage differentiation. Each MSC line was further characterized by its growth, proliferation, and viability as determined by cell count, bromodeoxyuridine incorporation, and cellular ATP levels, respectively. To determine whether these tests reliably predict the therapeutic aptitude of the MSCs, several lines were implanted in vivo to examine their capacity to engraft and form granulation tissue in a well-established murine wound model using polyvinyl alcohol sponges. Long-term engraftment of MSCs in the sponges was quantified through the presence of the human-specific Alu gene in sponge sections. Sections were also stained for proliferating cells, vascularity, and granulation tissue formation to determine successful engraftment and repair. We found that high performance in a combination of the in vitro tests accurately predicted which lines functioned well in vivo. These findings suggest that reliable and reproducible in vitro assays may be used to measure the functional potential of MSCs for therapeutic use.