Cerebral Creatine Deficiency Affects the Timing of Oligodendrocyte Myelination.

Cerebral creatine deficiency syndrome (CCDS) is an inborn error of metabolism characterized by intellectual delays, seizures, and autistic-like behavior. However, the role of endogenously synthesized creatine on CNS development and function remains poorly understood. Here, magnetic resonance spectroscopy of adult mouse brains from both sexes revealed creatine synthesis is dependent on the expression of the enzyme, guanidinoacetate methyltransferase (GAMT). To identify Gamt-expressed cells, and how Gamt affects postnatal CNS development, we generated a mouse line by knocking-in a GFP, which is expressed on excision of Gamt We found that Gamt is expressed in mature oligodendrocytes during active myelination in the developing postnatal CNS. Homozygous deletion of Gamt resulted in significantly reduced mature oligodendrocytes and delayed myelination in the corpus callosum. Moreover, the absence of endogenous creatine resulted in altered AMPK signaling in the brain, reduced brain creatine kinase expression in cortical neurons, and signs of axonal damage. Experimental demyelination in mice after tamoxifen-induced conditional deletion of Gamt in oligodendrocyte lineage cells resulted in delayed maturation of oligodendrocytes and myelin coverage in lesions. Moreover, creatine and cyclocreatine supplementation can enhance remyelination after demyelination. Our results suggest endogenously synthesized creatine controls the bioenergetic demand required for the timely maturation of oligodendrocytes during postnatal CNS development, and that delayed myelination and altered CNS energetics through the disruption of creatine synthesis might contribute to conditions, such as CCDS.SIGNIFICANCE STATEMENT Cerebral creatine deficiency syndrome is a rare disease of inborn errors in metabolism, which is characterized by intellectual delays, seizures, and autism-like behavior. We found that oligodendrocytes are the main source of endogenously synthesized creatine in the adult CNS, and the loss of endogenous creatine synthesis led to delayed myelination. Our study suggests impaired cerebral creatine synthesis affects the timing of myelination and may impact brain bioenergetics.

Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

NAD metabolism modulates inflammation and mitochondria function in diabetic kidney disease.

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United -States. Despite the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies, we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion, and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing the progression of diabetic kidney disease.

Estrogen-Related Receptor Agonism Reverses Mitochondrial Dysfunction and Inflammation in the Aging Kidney.

A gradual decline in renal function occurs even in healthy aging individuals. In addition to aging, per se, concurrent metabolic syndrome and hypertension, which are common in the aging population, can induce mitochondrial dysfunction and inflammation, which collectively contribute to age-related kidney dysfunction and disease. This study examined the role of the nuclear hormone receptors, the estrogen-related receptors (ERRs), in regulation of age-related mitochondrial dysfunction and inflammation. The ERRs were decreased in both aging human and mouse kidneys and were preserved in aging mice with lifelong caloric restriction (CR). A pan-ERR agonist, SLU-PP-332, was used to treat 21-month-old mice for 8 weeks. In addition, 21-month-old mice were treated with a stimulator of interferon genes (STING) inhibitor, C-176, for 3 weeks. Remarkably, similar to CR, an 8-week treatment with a pan-ERR agonist reversed the age-related increases in albuminuria, podocyte loss, mitochondrial dysfunction, and inflammatory cytokines, via the cyclic GMP-AMP synthase-STING and STAT3 signaling pathways. A 3-week treatment of 21-month-old mice with a STING inhibitor reversed the increases in inflammatory cytokines and the senescence marker, p21/cyclin dependent kinase inhibitor 1A (Cdkn1a), but also unexpectedly reversed the age-related decreases in PPARG coactivator (PGC)-1α, ERRα, mitochondrial complexes, and medium chain acyl coenzyme A dehydrogenase (MCAD) expression. These studies identified ERRs as CR mimetics and as important modulators of age-related mitochondrial dysfunction and inflammation. These findings highlight novel druggable pathways that can be further evaluated to prevent progression of age-related kidney disease.

Rise of the natural red pigment 'prodigiosin' as an immunomodulator in cancer.

Cancer is a heterogeneous disease with multifaceted drug resistance mechanisms (e.g., tumour microenvironment [TME], tumour heterogeneity, and immune evasion). Natural products are interesting repository of bioactive molecules, especially those with anticancer activities. Prodigiosin, a red pigment produced by Serratia marcescens, possesses inherent anticancer characteristics, showing interesting antitumour activities in different cancers (e.g., breast, gastric) with low or without harmful effects on normal cells. The present review discusses the potential role of prodigiosin in modulating and reprogramming the metabolism of the various immune cells in the TME, such as T and B lymphocytes, tumour-associated macrophages (TAMs), natural killer (NK) cells, and tumour-associated dendritic cells (TADCs), and myeloid-derived suppressor cells (MDSCs) which in turn might introduce as an immunomodulator in cancer therapy.

Optical and magnetic resonance imaging approaches for investigating the tumour microenvironment: state-of-the-art review and future trends.

The tumour microenvironment (TME) strongly influences tumorigenesis and metastasis. Two of the most characterized properties of the TME are acidosis and hypoxia, both of which are considered hallmarks of tumours as well as critical factors in response to anticancer treatments. Currently, various imaging approaches exist to measure acidosis and hypoxia in the TME, including magnetic resonance imaging (MRI), positron emission tomography and optical imaging. In this review, we will focus on the latest fluorescent-based methods for optical sensing of cell metabolism and MRI as diagnostic imaging tools applied both in vitro and in vivo. The primary emphasis will be on describing the current and future uses of systems that can measure intra- and extra-cellular pH and oxygen changes at high spatial and temporal resolution. In addition, the suitability of these approaches for mapping tumour heterogeneity, and assessing response or failure to therapeutics will also be covered.

Manuka honey enhanced sensitivity of HepG2, hepatocellular carcinoma cells, for Doxorubicin and induced apoptosis through inhibition of Wnt/ß-catenin and ERK1/2.

BACKGROUND: Recently, there is increasing awareness focused on the identification of naturally occurring anticancer agents derived from natural products. Manuka honey (MH) has been recognized for its biological properties as antimicrobial, antioxidant, and anticancer properties. However, its antiproliferative mechanism in hepatocellular carcinoma is not investigated. The current study focused mainly on investigating the molecular mechanism and synergistic effect of anticancer properties of MH on Doxorubicin (DOX)-mediated apoptotic cell death, using two different p53 statuses (HepG2 and Hep3B) and one non-tumorigenic immortalized liver cell line. RESULTS: MH treatment showed a proliferative inhibitory effect on tested cells in a dose-dependent manner with IC50 concentration of (6.92 ± 0.005%) and (18.62 ± 0.07%) for HepG2 and Hep3B cells, respectively, and induced dramatic morphological changes of Hep-G2 cells, which considered as characteristics feature of apoptosis induction after 48 h of treatment. Our results showed that MH or combined treatments induced higher cytotoxicity in p53-wild type, HepG2, than in p53-null, Hep3B, cells. Cytotoxicity was not observed in normal liver cells. Furthermore, the synergistic effect of MH and Dox on apoptosis was evidenced by increased annexin-V-positive cells and Sub-G1 cells in both tested cell lines with a significant increase in the percentage of Hep-G2 cells at late apoptosis as confirmed by the flow cytometric analysis. Consistently, the proteolytic activities of caspase-3 and the degradation of poly (ADP-ribose) polymerase were also higher in the combined treatment which in turn accompanied by significant inhibitory effects of pERK1/2, mTOR, S6K, oncogenic ß-catenin, and cyclin D1 after 48 h. In contrast, the MH or combined treatment-induced apoptosis was accompanied by significantly upregulated expression of proapoptotic Bax protein and downregulated expression of anti-apoptotic Bcl-2 protein after 48 h. CONCLUSIONS: Our data showed a synergistic inhibitory effect of MH on DOX-mediated apoptotic cell death in HCC cells. To our knowledge, the present study provides the first report on the anticancer activity of MH and its combined treatment with DOX on HCC cell lines, introducing MH as a promising natural and nontoxic anticancer compound.

Predicting new drug indications for prostate cancer: The integration of an in silico proteochemometric network pharmacology platform with patient-derived primary prostate cells.

BACKGROUND: Drug repurposing enables the discovery of potential cancer treatments using publically available data from over 4000 published Food and Drug Administration approved and experimental drugs. However, the ability to effectively evaluate the drug's efficacy remains a challenge. Impediments to broad applicability include inaccuracies in many of the computational drug-target algorithms and a lack of clinically relevant biologic modeling systems to validate the computational data for subsequent translation. METHODS: We have integrated our computational proteochemometric systems network pharmacology platform, DrugGenEx-Net, with primary, continuous cultures of conditionally reprogrammed (CR) normal and prostate cancer (PCa) cells derived from treatment-naive patients with primary PCa. RESULTS: Using the transcriptomic data from two matched pairs of benign and tumor-derived CR cells, we constructed drug networks to describe the biological perturbation associated with each prostate cell subtype at multiple levels of biological action. We prioritized the drugs by analyzing these networks for statistical coincidence with the drug action networks originating from known and predicted drug-protein targets. Prioritized drugs shared between the two patients' PCa cells included carfilzomib (CFZ), bortezomib (BTZ), sulforaphane, and phenethyl isothiocyanate. The effects of these compounds were then tested in the CR cells, in vitro. We observed that the IC50 values of the normal PCa CR cells for CFZ and BTZ were higher than their matched tumor CR cells. Transcriptomic analysis of CFZ-treated CR cells revealed that genes involved in cell proliferation, proteases, and downstream targets of serine proteases were inhibited while KLK7 and KLK8 were induced in the tumor-derived CR cells. CONCLUSIONS: Given that the drugs in the database are extremely well-characterized and that the patient-derived cells are easily scalable for high throughput drug screening, this combined in vitro and in silico approach may significantly advance personalized PCa treatment and for other cancer applications.

Divergent brain changes in two audiogenic rat strains: A voxel-based morphometry and diffusion tensor imaging comparison of the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR).

Acoustically evoked seizures (e.g., audiogenic seizures or AGS) are common in models of inherited epilepsy and occur in a variety of species including rat, mouse, and hamster. Two models that have been particularly well studied are the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR) strains. Acute and repeated AGS, as well as comorbid conditions, displays a close phenotypic overlap in these models. Whether these similarities arise from convergent or divergent structural changes in the brain remains unknown. Here, we examined the brain structure of Sprague Dawley (SD) and Wistar (WIS) rats, and quantified changes in the GEPR-3 and WAR, respectively. Brains from adult, male rats of each strain (n=8-10 per group) were collected, fixed, and embedded in agar and imaged using a 7 tesla Bruker MRI. Post-acquisition analysis included voxel-based morphometry (VBM), diffusion tensor imaging (DTI), and manual volumetric tracing. In the VBM analysis, GEPR-3 displayed volumetric changes in brainstem structures known to be engaged by AGS (e.g., superior and inferior colliculus, periaqueductal grey) and in forebrain structures (e.g., striatum, septum, nucleus accumbens). WAR displayed volumetric changes in superior colliculus, and a broader set of limbic regions (e.g., hippocampus, amygdala/piriform cortex). The only area of significant overlap in the two strains was the midline cerebellum: both GEPR-3 and WAR showed decreased volume compared to their control strains. In the DTI analysis, GEPR-3 displayed decreased fractional anisotropy (FA) in the corpus callosum, posterior commissure and commissure of the inferior colliculus (IC). WAR displayed increased FA only in the commissure of IC. These data provide a biological basis for further comparative and mechanistic studies in the GEPR-3 and WAR models, as well as provide additional insight into commonalities in the pathways underlying AGS susceptibility and behavioral comorbidity.

Neuropeptide Y as a Biomarker and Therapeutic Target for Neuroblastoma.

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)-mediated chemoresistance and Y2 receptor (Y2R)-mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.

Preclinical magnetic resonance imaging and systems biology in cancer research: current applications and challenges.

Biologically accurate mouse models of human cancer have become important tools for the study of human disease. The anatomical location of various target organs, such as brain, pancreas, and prostate, makes determination of disease status difficult. Imaging modalities, such as magnetic resonance imaging, can greatly enhance diagnosis, and longitudinal imaging of tumor progression is an important source of experimental data. Even in models where the tumors arise in areas that permit visual determination of tumorigenesis, longitudinal anatomical and functional imaging can enhance the scope of studies by facilitating the assessment of biological alterations, (such as changes in angiogenesis, metabolism, cellular invasion) as well as tissue perfusion and diffusion. One of the challenges in preclinical imaging is the development of infrastructural platforms required for integrating in vivo imaging and therapeutic response data with ex vivo pathological and molecular data using a more systems-based multiscale modeling approach. Further challenges exist in integrating these data for computational modeling to better understand the pathobiology of cancer and to better affect its cure. We review the current applications of preclinical imaging and discuss the implications of applying functional imaging to visualize cancer progression and treatment. Finally, we provide new data from an ongoing preclinical drug study demonstrating how multiscale modeling can lead to a more comprehensive understanding of cancer biology and therapy.

Alterations in glucose homeostasis in a murine model of Chagas disease.

Chagas disease, caused by Trypanosoma cruzi, is an important cause of morbidity and mortality primarily resulting from cardiac dysfunction, although T. cruzi infection results in inflammation and cell destruction in many organs. We found that T. cruzi (Brazil strain) infection of mice results in pancreatic inflammation and parasitism within pancreatic ß-cells with apparent sparing of α cells and leads to the disruption of pancreatic islet architecture, ß-cell dysfunction, and surprisingly, hypoglycemia. Blood glucose and insulin levels were reduced in infected mice during acute infection and insulin levels remained low into the chronic phase. In response to the hypoglycemia, glucagon levels 30 days postinfection were elevated, indicating normal α-cell function. Administration of L-arginine and a ß-adrenergic receptor agonist (CL316, 243, respectively) resulted in a diminished insulin response during the acute and chronic phases. Insulin granules were docked, but the lack of insulin secretion suggested an inability of granules to fuse at the plasma membrane of pancreatic ß-cells. In the liver, there was a concomitant reduced expression of glucose-6-phosphatase mRNA and glucose production from pyruvate (pyruvate tolerance test), demonstrating defective hepatic gluconeogenesis as a cause for the T. cruzi-induced hypoglycemia, despite reduced insulin, but elevated glucagon levels. The data establishes a complex, multi-tissue relationship between T. cruzi infection, Chagas disease, and host glucose homeostasis.

ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Response of adipose tissue to early infection with Trypanosoma cruzi (Brazil strain).

Brown adipose tissue (BAT) and white adipose tissue (WAT) and adipocytes are targets of Trypanosoma cruzi infection. Adipose tissue obtained from CD-1 mice 15 days after infection, an early stage of infection revealed a high parasite load. There was a significant increase in macrophages in infected adipose tissue and a reduction in lipid accumulation, adipocyte size, and fat mass and increased expression of lipolytic enzymes. Infection increased levels of Toll-like receptor (TLR) 4 and TLR9 and in the expression of components of the mitogen-activated protein kinase pathway. Protein and messenger RNA (mRNA) levels of peroxisome proliferator-activated receptor γ were increased in WAT, whereas protein and mRNA levels of adiponectin were significantly reduced in BAT and WAT. The mRNA levels of cytokines, chemokines, and their receptors were increased. Nuclear Factor Kappa B levels were increased in BAT, whereas Iκκ-γ levels increased in WAT. Adipose tissue is an early target of T. cruzi infection.

Amniotic stem cells as a source of regenerative medicine to treat female infertility.

Impaired reproductive health is a worldwide problem that affects the psychological well-being of a society. Despite the technological developments to treat infertility, the global infertility rate is increasing significantly. Many infertility conditions are currently treated using various advanced clinical approaches such as intrauterine semination (IUI), in vitro fertilization (IVF), and intracytoplasmic injection (ICSI). Nonetheless, clinical management of some conditions such as dysfunctional endometrium, premature ovarian failure, and ovarian physiological aging still pose significant challenges. Stem cells based therapeutic strategies have a long-standing history to treat many infertility conditions, but ethical restrictions do not allow the broad-scale utilization of adult mesenchymal stromal/stem cells (MSCs). Easily accessible, placental derived or amniotic stem cells present an invaluable alternative source of non-immunogenic and non-tumorigenic stem cells that possess multilineage potential. Given these characteristics, placental or amniotic stem cells (ASCs) have been investigated for therapeutic purposes to address infertility in the last decade. This study aims to summarize the current standing and progress of human amniotic epithelial stem cells (hAECs), amniotic mesenchymal stem cells (hAMSCs), and amniotic fluid stem cells (hAFSCs) in the field of reproductive medicine. The therapeutic potential of these cells to restore or enhance normal ovarian function and pregnancy outcomes are highlighted in this study.

Provocative non-canonical roles of p53 and AKT signaling: A role for Thymosin ß4 in medulloblastoma.

The PI3K/AKT and p53 pathways are key regulators of cancer cell survival and death, respectively. Contrary to their generally accepted roles, several lines of evidence, including ours in medulloblastoma, the most common childhood brain cancer, highlight non-canonical functions for both proteins and show a complex context-dependent dynamic behavior in determining cell fate. Interestingly, p53-mediated cell survival and AKT-mediated cell death can dominate in certain conditions, and these interchangeable physiological functions may potentially be manipulated for better clinical outcomes. This review article presents studies in which p53 and AKT behave contrary to their well-established functions. We discuss the factors and circumstances that may be involved in mediating these changes and the implications of these unique roles of p53 and AKT in devising therapeutic strategies. Lastly, based on our recent finding of Thymosin beta 4-mediated chemosensitivity via an AKT-p53 interaction in medulloblastoma cells, we also discuss the possible implications of Thymosin beta-4 in enhancing drug sensitivity in this deadly childhood disease.

Expansion of human amniotic epithelial cells using condition cell reprogramming technology.

Human amniotic epithelial cells (hAECs) are non-immunogenic epithelial cells that can develop into cells of all three germline lineages. However, a refined clinically reliable method is required to optimize the preparation and banking procedures of hAECs for their successful translation into clinical studies. With the goal of establishing standardized clinically applicable hAECs cultured cells, we described the use of a powerful epithelial cell culture technique, termed Conditionally Reprogrammed Cells (CRC) for ex vivo expansion of hAECs. The well-established CRC culture method uses a Rho kinase inhibitor (Y-27632) and J2 mouse fibroblast feeder cells to drive the indefinite proliferation of all known epithelial cell types. In this study, we used an optimized CRC protocol to successfully culture hAECs in a CRC medium supplemented with xenogen-free human serum. We established that hAECs thrive under the CRC conditions for over 5 passages while still expressing pluripotent stem markers (OCT-4, SOX-2 and NANOG) and non-immunogenic markers (CD80, CD86 and HLA-G) suggesting that even late-passage hAECs retain their privileged phenotype. The hAECs-CRC cells were infected with a puromycin-selectable lentivirus expressing luciferase and GFP (green fluorescent protein) and stably selected with puromycin. The hAECs expressing GFP were injected subcutaneously into the flanks of Athymic and C57BL6 mice to check the tolerability and stability of cells against the immune system. Chemiluminescence imaging confirmed the presence and viability of cells at days 2, 5, and 42 without acute inflammation or any tumor formation. Collectively, these data indicate that the CRC approach offers a novel solution to expanding hAECs in humanized conditions for future clinical uses, while retaining their primary phenotype.

Recent developments in the interactions between caveolin and pathogens.

The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.

A comparison of isolation and culture protocols for human amniotic mesenchymal stem cells.

The successful translation of mesenchymal stem cells (MSCs) from bench to bedside is predicated upon their regenerative capabilities and immunomodulatory potential. Many challenges still exist in making MSCs a viable and cost-effective therapeutic option, due in part to the challenges of sourcing MSCs from adult tissues and inconsistencies in the characterization of MSCs. In many cases, adult MSC collection is an invasive procedure, and ethical concerns and age-related heterogeneity further complicate obtaining adult tissue derived MSCs at the scales needed for clinical applications. Alternative adult sources, such as post-partum associated tissues, offer distinct advantages to overcome these challenges. However, successful therapeutic applications rely on the efficient ex-vivo expansion of the stem cells while avoiding any culture-related phenotypic alterations, which requires optimized and standardized isolation, culture, and cell preservation methods. In this review, we have compared the isolation and culture methods for MSCs originating from the human amniotic membrane (hAMSCs) of the placenta to identify the elements that support the extended subculture potential of hAMSCs without compromising their immune-privileged, pluripotent regenerative potential.Abbreviations: AM: Human amniotic membrane; ASCs: Adipose tissue-derived stem cells; BM-MSCs: Bone marrow-mesenchymal stem cells; DMEM: Dulbecco's modified eagle medium; DT: Doubling time; EMEM: Eagle's modified essential medium; ESCM: Embryonic stem cell markers; ESCs: Embryonic stem cells; hAECs: Human amniotic epithelial cells; hAMSCs: Human amniotic mesenchymal stem cells; HLA: Human leukocyte antigen; HM: Hematopoietic markers; IM: Immunogenicity markers; MHC: Major histocompatibility complex; MSCs: Mesenchymal stem cells; MCSM: Mesenchymal cell surface markers; Nanog: NANOG homeobox; Oct: Octamer binding transcription factor 4; P: Passage; PM: Pluripotency markers; STRO-1: Stromal precursor antigen-1; SCP: Subculture potential; Sox-2: Sry-related HMG box gene 2; SSEA-4: Stage-specific embryonic antigen; TRA: Tumor rejection antigen.

A Survey and Critical Evaluation of Isolation, Culture, and Cryopreservation Methods of Human Amniotic Epithelial Cells.

Human amniotic epithelial cells (hAECs), derived from an epithelial cell layer of the human amniotic membrane, possess embryonic stem-like properties and are known to maintain multilineage differentiation potential. Unfortunately, an inability to expand hAECs without significantly compromising their stem cell potency has precluded their widespread use for regenerative therapies. This article critically evaluates the methods used for isolation, expansion, and cryopreservation of hAECs. We assessed the impact of these methods on ex-vivo expansion and stem cell phenotype of hAECs. Moreover, the progress and challenges to optimize clinically suitable culture conditions for an efficient ex-vivo expansion and storage of these cells are highlighted. Additionally, we also reviewed the currently used hAECs isolation and characterization methods employed in clinical trials. Despite the developments made in the last decade, significant challenges still exist to overcome limitations of ex-vivo expansion and retention of stemness of hAECs in both xenogeneic and xenofree culture conditions. Therefore, optimization and standardization of culture conditions for robust ex-vivo maintenance of hAECs without affecting tissue regenerative properties is an absolute requirement for their successful therapeutic manipulation. This review may help the researchers to optimize the methods that support ex-vivo survival, proliferation, and self-renewal properties of the hAECs.Abbreviations: AM: Human amniotic membrane; CM-HBSS: Ca++ and Mg++ free HBSS; DMEM: Dulbecco's Modified Eagle Medium; DMEM-HG: DMEM-high glucose; EMEM: Eagle's Modified Essential Medium; EMT: Epithelial-to-mesenchymal transition; EpM: Epi-life complete media; ESC: Embryonic stem cells; ESCM: Epithelial cell surface markers; hAECs: Human amniotic epithelial cells; HLA: Human leukocyte antigen; IM: Immunogenicity markers; iPSC: Induced pluripotent stem cells; KOSR; KSR: Knockout serum replacement; KSI: Key success indicators; CHM: Cell heterogeneity markers; Nanog: NANOG homeobox; Oct-4: Octamer binding transcription factor 4; OR: Operation room; P: Passage; PM: Pluripotency markers; SCM: Stem cell markers for non-differentiated cells; Sox-2: Sry-related HMG box gene 2; SSEA-4: Stage-specific embryonic antigen; TRA: Tumor rejection antigen; UC: Ultra-culture; XF: Xenogeneic free.