Extraintestinal Manifestation of Yersinia pseudotuberculosis Bacteremia as Acute Hepatitis: Case Report and Review of the Literature.

Yersinia pseudotuberculosis is a causative agent of foodborne zoonosis that usually causes self-limiting pseudoappendicitis. Y. pseudotuberculosis infection also causes systemic spread or extraintestinal manifestations in patients with predisposing conditions. Here, we present a case of acute hepatitis with Y. pseudotuberculosis bacteremia in a 30-year-old man. He was previously healthy without significant medical history other than obesity and current smoking. At the time of admission, he presented with high fever accompanied by chills, jaundice, abdominal pain, and watery diarrhea. Laboratory studies revealed leukocytosis and elevated liver function parameters. A stool culture showed no causative pathogens. Empiric antibiotic therapy with ceftriaxone and metronidazole was administered. Y. pseudotuberculosis was later isolated from the initial blood culture performed on the day of admission using MALDI-TOF mass spectrometry. Antibiotic treatment was continued based on the susceptibility testing results from MALDI-TOF MS and VITEk®2, as well as clinical and laboratory improvements. The patient was discharged on the tenth day of admission and remained healthy with no recurrence during the 12-month follow-up. Here, we review the literature on the systemic infection caused by Y. pseudotuberculosis, including extraintestinal manifestations. This case highlights that Y. pseudotuberculosis may be considered a differential causative organism in patients with acute colitis and hepatitis.

Analysis of bone marrow supernatant neutrophil gelatinase-associated lipocalin and hematological parameters in hematological malignancy.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a urine biomarker related to acute renal injury. Whereas several studies have evaluated NGAL levels in hematological malignancy, using peripheral blood (PB). Recently, bone marrow (BM) NGAL level was reported to be higher than PB NGAL level in individuals with hematological malignancy, suggesting that BM NGAL would reflect BM microenvironment better than PB NGAL. We measured BM NGAL levels in patients with hematological malignancy, comparing those with NGAL levels in normal BM. We evaluated the association of BM NGAL with hematological parameters including neutrophil counts. METHODS: BM samples were collected from 107 patients who underwent BM examination. Immunoassays were used to assess NGAL levels. Data on hematological parameters were collected from medical records. Intergroup comparisons were performed using the Kruskal-Wallis H test and Pearson chi-square test. Single and multiple regression analyses were performed to analyze the relationships. RESULTS: The independent factors that affected the BM NGAL level were neutrophil counts and BM band neutrophil%, while neutrophil count was the main influencing factor. The acute myeloid leukemia (n = 18) and myelodysplastic syndrome (n = 25) groups showed statistically lower BM NGAL levels than patients with normal BM. The myeloproliferative neoplasm group (n = 34) showed higher BM NGAL levels than patients with normal BM, but this difference was not statistically significant. Neutrophil counts and BM band neutrophil% showed intergroup patterns similar to those of BM NGAL levels. CONCLUSION: BM NGAL was related to neutrophil count and BM band neutrophil%, showing different levels according to hematological malignant disease entities.

Distinct and Shared Determinants of Cardiomyocyte Contractility in Multi-Lineage Competent Ethnically Diverse Human iPSCs.

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics, epigenomics, and bioinformatics that inform on genetic pathways directing tissue development and function. When possible, population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American, Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype, verified teratoma formation, pluripotency biomarkers, and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural, pancreatic, and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential.

Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines.

The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair, and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian, and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations.

Caudalized human iPSC-derived neural progenitor cells produce neurons and glia but fail to restore function in an early chronic spinal cord injury model.

Neural progenitor cells (NPCs) have shown modest potential and some side effects (e.g. allodynia) for treatment of spinal cord injury (SCI). In only a few cases, however, have NPCs shown promise at the chronic stage. Given the 1.275 million people living with chronic paralysis, there is a significant need to rigorously evaluate the cell types and methods for safe and efficacious treatment of this devastating condition. For the first time, we examined the pre-clinical potential of NPCs derived from human induced pluripotent stem cells (hiPSCs) to repair chronic SCI. hiPSCs were differentiated into region-specific (i.e. caudal) NPCs, then transplanted into a new, clinically relevant model of early chronic cervical SCI. We established the conditions for successful transplantation of caudalized hiPSC-NPCs and demonstrate their remarkable ability to integrate and produce multiple neural lineages in the early chronic injury environment. In contrast to prior reports in acute and sub-acute injury models, survival and integration of hiPSC-derived neural cells in the early chronic cervical model did not lead to significant improvement in forelimb function or induce allodynia. These data indicate that while hiPSCs show promise, future work needs to focus on the specific hiPSC-derivatives or co-therapies that will restore function in the early chronic injury setting.

Downregulation of H19 improves the differentiation potential of mouse parthenogenetic embryonic stem cells.

Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.

Generation of leukemia inhibitory factor and basic fibroblast growth factor-dependent induced pluripotent stem cells from canine adult somatic cells.

For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases.

Mitochondrial rejuvenation after induced pluripotency.

BACKGROUND: As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion "resets" some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal. CONCLUSIONS/SIGNIFICANCE: These results - coupled with earlier data from our laboratory - suggest that IPSC conversion not only resets the "biological clock", but can also rejuvenate the energetic capacity of derived cells.

Human-induced pluripotent stem cells produced under xeno-free conditions.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.

Telomere dynamics in human cells reprogrammed to pluripotency.

BACKGROUND: Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell "rejuvenation." METHODOLOGY/PRINCIPAL FINDINGS: We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres. CONCLUSIONS/SIGNIFICANCE: While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.

Dynamic epigenetic regulation of the Oct4 and Nanog regulatory regions during neural differentiation in rhesus nuclear transfer embryonic stem cells.

Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog transcripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were methylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic enrichment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the parameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.