Anti-alpha Fetoprotein Anticorpo [AFP-01] (A85533)

Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

A simple, scalable, and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i), or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel, we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers, confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs).

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive, result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs, we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate, formulated as a hypertonic solution, gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium, retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together, this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology, high levels (>80%) of pluripotency markers OCT4, SSEA-4, TRA-1-60 andTRA-1-81, a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers, both in vivo and in vitro.

BACKGROUND:

MicroRNA-630 (miR-630) has been shown to be involved in various human malignancies. However, its role in hepatocellular carcinoma (HCC) remains unknown.

MATERIAL AND METHODS:

TaqMan qRT-PCR assay was performed to detect the expression of miR-630 in 42 pairs of HCC tissues and corresponding noncancerous hepatocellular tissues, and its correlations with clinicopathologic features and serum alpha-fetoprotein (AFP) level of patients were analyzed.

RESULTS:

The present study found that miR-630 expression was significantly increased in HCC tissues and cells compared with their normal counterparts. miR-630 expression level did not significantly chang at stage I but was markedly increased at advanced TNM stage (stage II~III). In addition, the increased expression of miR-630 in tissues of HCC appeared in patients who exhibited elevated serum levels of AFP (>25 ng/ml), but not in those with normal AFP levels (≤25 ng/ml). The miR-630 expression in carcinoma tissues revealed a positive correlation with the levels of serum alpha-fetoprotein (AFP; R2=0.768).

CONCLUSIONS:

These results suggest that miR-630 is associated with tumor progression of hepatocellular carcinoma and may be a potential prognosis indicator.

BACKGROUND:

Exposures of pregnant women to natural and manmade chemicals can lead to negative health effects in the baby, ranging from low birth weight to developmental defects. In some cases, diseases were postulated to have their basis in toxic exposure in utero or in early childhood. Therefore, an understanding of fetal responses to environmental exposures is essential. To that end, cord blood is a readily accessible biofluid whose proteomic makeup remains mostly unexplored when compared with that of adults.

OBJECTIVES:

Our goal was an initial global assessment of the fetal serum proteome and for the identification of protein biomarkers indicative of toxic in utero exposures related to maternal cigarette smoking.

METHODS:

Drawing from a repository of 300 samples, we selected umbilical cord blood sera from 12 babies born to six smokers and six nonsmokers and analyzed both sample pools by tandem mass spectrometry in conjunction with isobaric tags (iTRAQ) for protein quantification.

RESULTS:

We identified 203 proteins, 17 of which were differentially expressed between the cigarette smoke-exposed and control populations. Most of the identified candidate biomarkers were biologically plausible, thereby underscoring the feasibility of screening neonates with global proteomic techniques for biomarkers of exposure and early biological effects triggered by in utero chemical exposures.

CONCLUSIONS:

This validation study provides an initial view of the proteome of human cord blood sera; it demonstrates the feasibility of identifying therein by use of proteomics, biomarkers of environmental, toxic exposures.

Current hypothesis suggest that tumors can originate from adult cells after a process of 'reprogramming' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic 'Warburg' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that 'in vitro' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate 'stem' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate.

Surface-enhanced Raman scattering (SERS) spectroscopy possesses the most promising advantage of multiplex detection for biosensing applications, which is achieved due to the narrow 'fingerprint' Raman spectra from the analyte molecules. We developed an ultrasensitive platform for the multiplex detection of cancer biomarkers by combining the SERS technique with a hollow-core photonic crystal fiber (HCPCF). Axially aligned air channels inside the HCPCF provide an excellent platform for optical sensing using SERS. In addition to the flexibility of optical fibers, HCPCF provides better light confinement and a larger interaction length for the guided light and the analyte, resulting in an improvement in sensitivity to detect low concentrations of bioanalytes in extremely low sample volumes. Herein, for the first time, we demonstrate the sensitive multiplex detection of biomarkers immobilized inside the HCPCF using antibody-conjugated SERS-active nanoparticles (SERS nanotags). As a proof-of-concept for targeted multiplex detection, initially we carried out the sensing of epidermal growth factor receptor (EGFR) biomarker in oral squamous carcinoma cell lysate using three different SERS nanotags. Subsequently, we also achieved simultaneous detection of hepatocellular carcinoma (HCC) biomarkers-alpha fetoprotein (AFP) and alpha-1-antitrypsin (A1AT) secreted in the supernatant from Hep3b cancer cell line. Using a SERS-HCPCF sensing platform, we could successfully demonstrate the multiplex detection in an extremely low sample volume of ∼20 nL. In future, this study may lead to sensitive biosensing platform for the low concentration detection of biomarkers in an extremely low sample volume of body fluids to achieve early diagnosis of multiple diseases. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Nuclear factor-kappaB (NF-kappaB) plays a critical role during fetal liver development and hepatic oncogenesis. Here, we have assessed whether NF-kappaB activity is required for murine hepatocellular carcinoma cell survival. We show that adenoviral-mediated inhibition of inhibitor of NF-kappaB kinase-beta (IKK-2) activity in hepatocellular carcinomas derived from transforming growth factor (TGF)-alpha/c-myc bitransgenic mice leads to inhibition of NF-kappaB and promotes tumor necrosis factor (TNF)-alpha-mediated cell death of malignant hepatocytes but not the surrounding peritumorous tissue. Induction of apoptosis is accompanied by inhibition of Bcl-X(L) and XIAP, two pro-survival NF-kappaB target genes. In addition, we have identified the alpha-fetoprotein (AFP) as a novel downstream target of NF-kappaB. We show that repression of IKK-2 activity in hepatocellular carcinomas promotes down-regulation of AFP gene expression. Likewise, genetic disruption of the RelA subunit results in reduced AFP gene expression during embryonic liver development, at a time in which fetal hepatocytes are sensitized to TNF-alpha-mediated cell killing. In this regard, we show that AFP inhibits TNF-alpha-induced cell death of murine hepatocellular carcinomas through association with TNF-alpha and inhibition of TNFRI signaling. Thus, NF-kappaB-mediated regulation of AFP gene expression during liver tumor formation and embryonic development of the liver constitutes a potential novel mechanism used by malignant and fetal hepatocytes to evade immune surveillance.

Seven murine hybridomas were established producing IgG1 monoclonal antibodies (mAbs) against three different epitopes of human alpha-fetoprotein (alpha FP). The interaction of two of these mAbs (AFP-01 and AFP-02) with the antigen was strongly inhibited by calcium-chelating agents. The mAb AFP-11 could be used for estimation of alpha FP by RIA in the concentration range of 5-100 ng/ml. Several combinations of the mAbs could be used in a two-site sandwich-ELISA for similar purposes. Immunoaffinity chromatography on a column of immobilized mAb AFP-01 permitted the purification of alpha FP.