Integrative Analysis of Colonic Biopsies from Inflammatory Bowel Disease Patients Identifies an Interaction Between Microbial Bile Acid-inducible Gene Abundance and Human Angiopoietin-like 4 Gene Expression.

BACKGROUND AND AIMS: Microbial-derived bile acids can modulate host gene expression, and their faecal abundance is decreased in active inflammatory bowel disease [IBD]. We analysed the impact of endoscopic inflammation on microbial genes involved in bile acid biotransformation, and their interaction with host transcriptome in the intestinal mucosa of IBD patients. METHODS: Endoscopic mucosal biopsies were collected from non-inflamed and inflamed terminal ileum, ascending and sigmoid colon of IBD patients. Prediction of imputed metagenome functional content from 16S rRNA profile and real-time quantitative polymerase chain reaction [qPCR] were utsed to assess microbial bile acid biotransformation gene abundance, and RNA-seq was used for host transcriptome analysis. Linear regression and partial Spearman correlation accounting for age, sex, and IBD type were used to assess the association between microbial genes, inflammation, and host transcriptomics in each biopsy location. A Bayesian network [BN] analysis was fitted to infer the direction of interactions between IBD traits and microbial and host genes. RESULTS: The inferred microbial gene pathway involved in secondary bile acid biosynthesis [ko00121 pathway] was depleted in inflamed terminal ileum of IBD patients compared with non-inflamed tissue. In non-inflamed sigmoid colon, the relative abundance of bile acid-inducible [baiCD] microbial genes was positively correlated with the host Angiopoietin-like 4 [Angptl4] gene expression. The BN analysis suggests that the microbial baiCD gene abundance could affect Angptl4 expression, and this interaction appears to be lost in the presence of inflammation. CONCLUSIONS: Endoscopic inflammation affects the abundance of crucial microbial bile acid-metabolising genes and their interaction with Angptl4 in intestinal mucosa of IBD patients.

Human umbilical cord perivascular cells maintain regenerative traits following exposure to cyclophosphamide.

Chemotherapies can cause germ cell depletion and gonadal failure. When injected post-chemotherapy, mesenchymal stromal cells (MSCs) from various sources have been shown to have regenerative effects in rodent models of chemotherapy-induced gonadal injury. Here, we evaluated two properties of a novel source of MSC, first trimester (FTM) human umbilical cord perivascular cells (HUCPVCs) (with increased regenerative potential compared to older sources), that may render them a promising candidate for chemotherapeutic gonadal injury prevention. Firstly, their ability to resist the cytotoxic effects of cyclophosphamide (CTX) in vitro, as compared to term HUCPVCs and bone marrow cells (BMSCs); and secondly, whether they prevent gonadal dysfunction if delivered prior to gonadotoxic therapy in vivo. BMSC, FTM HUCPVC, term HUCPVC, and control NTERA2 cells were treated with moderate (150 µmol/L) and high (300 µmol/L) doses of CTX in vitro. Viability, proliferative capacity, mesenchymal cell lineage markers and differentiation capacity, immunogenicity, and paracrine gene expression were assessed. CTX was administered to Wistar rats 2 days following an intra-ovarian injection of FTM HUCPVC. HUCPVC survival and ovarian follicle numbers were assessed using histological methods. We conclude that FTM HUCPVC maintain key regenerative properties following chemotherapy exposure and that pre-treatment with these cells may prevent CTX-induced ovarian damage in vivo. Therefore, HUCPVCs are promising candidates for fertility preservation.

Human umbilical cord perivascular cells prevent chemotherapeutic drug-induced male infertility in a mouse model.

OBJECTIVE: To study whether intratesticular (IT) administration of 2 sources of human umbilical cord perivascular cells (HUCPVC), rich and potent sources of mesenchymal stromal cells (MSC), before chemotherapy can prevent infertility in a mouse model. DESIGN: Two control groups of CD1 male mice without busulfan (BUS) administration (untreated and IT media injection groups) were included. Experimental groups included IT administration of media, first trimester (FTM) HUCPVCs or term HUCPVCs (n = 5 each) injected 3 days before BUS treatment (20 mg/kg). All groups were included in a mating time course study over 6 months. SETTING: Preclinical study in a fertility center research laboratory. PATIENTS: Not applicable. INTERVENTION: IT delivery of FTM or term HUCPVC before BUS treatment. MAIN OUTCOME MEASURES: Pregnancies, litter sizes, and gross morphology of offspring were monitored. Caudal epididymal sperm concentration, motility, and progressive motility were assessed by computer-assisted sperm analysis. Spermatogenesis was also assessed histologically in testicular tissue sections. RESULTS: FTM and term HUCPVC displayed an MSC-associated immunophenotype and expressed transcripts encoding paracrine factors known to regulate the testicular cell niche. IT administration of FTM and term HUCPVC before chemotherapy promoted the recovery of spermatogenesis and fertility compared with BUS-treated animals that received a media injection. Although the total number of pups sired over 6 months by males treated with FTM or term HUCPVC was reduced compared with untreated or media-injected controls, litter size and sperm parameters in fertile animals did not differ between control and cell-treated groups. CONCLUSION: HUCPVC represent a promising source of MSC-based therapy to prevent gonadotoxic chemotherapeutic drug-induced infertility.

Differential Expression of microRNAs in Peripheral Blood Mononuclear Cells Identifies Autophagy and TGF-Beta-Related Signatures Aberrantly Expressed in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: MicroRNAs [miRNAs] have emerged as important regulators in inflammatory bowel disease [IBD]. This study investigated differential expression of miRNAs across clinical phenotypes in a well-characterized cohort of IBD patients and healthy controls [HCs]. METHODS: A cohort of Crohn's disease [CD] and ulcerative colitis [UC] patients and HCs was prospectively accrued. Total RNA was extracted from peripheral blood mononuclear cells for all subjects. miRNA expression was measured using NanoString technologies. The subjects were stratified according to disease activity and location. Statistical significance was assessed per miRNA across outcomes and corrected for multiple testing. miRNA regulation of transcription of important results was confirmed in vitro by a dual luciferase reporter assay and autophagy function was evaluated using immunofluorescence imaging of LC3 puncta in HeLa cells. RESULTS: In total, 120 subjects were enrolled. Seventy-four miRNAs were differentially expressed across CD, UC and HCs. Comparing quiescent CD [CDq] with HCs we found ten miRNAs upregulated in CDq. When comparing colonic CD [CCD] to UC, seven miRNAs were upregulated in CCD. The most differentially expressed miRNA in CCD vs UC was miR-874-3p, and we showed its possible utility as a biomarker of differential diagnosis. We showed miR-874-3p targets ATG16L1 and reduces its expression in vitro. An miR-874-3p mimic dysregulates autophagy by a reduction of LC3 in vitro. CONCLUSIONS: We identified unique miRNA signatures expressed in distinct IBD phenotypes. These associations highlight pathways dysregulated by aberrant miRNA expression, revealing possible mechanisms underlying the pathophysiology of IBD, but also suggest a cluster of miRNAs as readily accessible biomarkers to aid in differential diagnosis.

Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro.

Spermatogonial Stem Cell (SSC) expansion in vitro remains a major challenge in efforts to preserve fertility among pubertal cancer survivor boys. The current study focused on innovative approaches to optimize SSC expansion. Six- to eight-week-old CD-1 murine testicular samples were harvested by mechanical and enzymatic digestion. Cell suspensions were incubated for differential plating (DP). After DP, we established two experiments comparing single vs. repetitive DP (S-DP and R-DP, respectively) until passage 2 (P2) completion. Each experiment included a set of cultures consisting of 5 floating-to-attached cell ratios (5, 10, 15, 20, and 25) and control cultures containing floating cells only. We found similar cell and colony count drops during P0 in both S- and R-DP. During P2, counts increased in S-DP in middle ratios (10, 15, and especially 20) relative to low and high ratios (5 and 25, respectively). Counts dropped extensively in R-DP after passage 2. The superiority of intermediate ratios was demonstrated by enrichment of GFRα1 by qPCR. The optimal ratio of 20 in S-DP contained significantly increased proportions of GFRα1-positive cells (25.8±5.8%) as measured by flow cytometry compared to after DP (1.9±0.7%, p<0.0001), as well as positive immunostaining for GFRα1 and UTF1, with rare Sox9-positive cells. This is the first report of the impact of initial floating-to-attached cell ratios on SSC proliferation in vitro. ABBREVIATIONS: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting.

Human umbilical perivascular cells: a novel source of MSCs to support testicular niche regeneration.

The expansion of functional testicular biopsy-derived human spermatogonial stem cells (hSSC) ex-vivo may enable the restoration of fertility in pre-pubertal males having undergone gonadotoxic therapies or men with severe male factor infertility. Various somatic cells are known to regulate SSC homeostasis and spermatogenesis in the developing and adult testis. Prior attempts to recapitulate this niche demonstrated the requirement of feeder cells, such as endogenous testicular somatic cells, for germ cell expansion ex-vivo. However, this strategy has limitations for the expansion of hSSCs from tissue biopsies where spermatogenesis is absent or defective. Our aim was to evaluate first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel source of mesenchymal stromal-like cells (MSCs), as potential human feeder cells for standardized hSSC expansion ex-vivo. Targeted RNA sequencing analysis demonstrated that CD90+ve FTM HUCPVCs expanded in vitro under germ cell culture conditions express a profile of targeted testicular-associated transcripts that is similar to cultured human CD90+ve testicular adherent cells (hTACs) and secrete LIF, FGF2 and BMP4, key growth factors known to regulate spermatogenesis. We also demonstrated that mitotically-inactivated FTM HUCPVCs support the expansion of mouse germ cells and putative SSCs ex-vivo, and that FTM HUCPVC transplantation promotes in vivo germ cell regeneration following mono-2- ethylhexyl phthalate (MEHP)-induced seminiferous tubule damage in a murine model, including a partial reconstitution of tubular cellular architecture and reestablishment of DAZL and acrosin+ve germ cell layers. Together, these data suggest that FTM HUCPVCs have phenotypical and functional properties that may support repair of the human testicular niche.

Somatic cells, stem cells, and induced pluripotent stem cells: how do they now contribute to conservation?

More than a decade has now passed since the birth of the first endangered species produced from an adult somatic cell reprogrammed by somatic cell nuclear transfer. At that time, advances made in domestic and laboratory animal species provided the necessary foundation for attempting cutting-edge technologies on threatened and endangered species. In addition to nuclear transfer, spermatogonial stem cell transplantation and induction of pluripotent stem cells have also been explored. Although many basic scientific questions have been answered and more than 30 wild species have been investigated, very few successes have been reported. The majority of studies document numerous obstacles that still need to be overcome to produce viable gametes or embryos for healthy offspring production. This chapter provides an overview of somatic cell and stem cell technologies in different taxa (mammals, fishes, birds, reptiles and amphibians) and evaluates the potential and impact of these approaches for animal species conservation.

Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies.

The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary.