Aurora B kinase (AURKB) and epidermal growth factor receptor 1 (EGFR) belong to serine/threonine and tyrosine kinase family of proteins. Both these kinases are found to overexpress in a large number of epithelial cancers, including hormonal refractory prostate cancer. In this communication, we present evidence for down-regulated expression of AURKB and EGFR, either alone or in combination, in prostate cancer cells. The results show that AURKB and EGFR were efficiently down-regulated in a dose-dependent manner. AURKB and EGFR siRNA in combination have shown enhanced therapeutic effect by inhibiting PC3 cell proliferation and inducing apoptosis in vitro, whereas androgen-dependent cancer cells LNCaP remain unaffected correlating the endogenous expression levels. Knockdown of AURKB and EGFR individually resulted in inhibition of prostate tumor growth in athymic nude mice and their combined knockdown resulted in tumor regression in which 40% of the treated mice were found to be tumor free, implicating the potential therapeutic benefits of AURKB-EGFR combination therapy.
Our study demonstrates that BMMSCs can be transdifferentiated efficiently into functional dopaminergic neurons both in vitro and in vivo. This holds immense clinical potential as a replacement therapy for PD and other neurodegenerative diseases.
We describe a method of generating an enriched population of NCAM-positive cells from a human teratocarcinoma cell line (NTera2/D1) and their differentiation into midbrain dopaminergic neurons in the absence of the caudalizing factor retinoic acid (RA). NTera2 cells were induced to form embryoid bodies and then to generate nestin-positive cells on treatment with serum-free defined medium supplemented with neurotrophic factors. We enriched the neuroprogenitor population by magnetic sorting of the nestin-positive cells using the antibody to neural cell adhesion molecule (NCAM). These cells were expanded by exposing them to the signaling molecule sonic hedgehog (SHH) in conjunction with fibroblast growth factor-8 (FGF-8). The predifferentiated cells when analyzed by RT-PCR showed expression of dopaminergic markers such as Nurr1, Engrailed-1, aromatic amino decarboxylase (AADC), VMAT2, tyrosine hydroxylase (TH), and dopamine transporter (DAT). These cells also stained positively for protein markers such as nestin, NCAM, MAP-2, and TH. We further demonstrated that when transplanted into the brain of Parkinsonian rats, these neuroprogenitor cells did not form tumors but differentiated into dopaminergic neurons, as revealed by TH immunolabeling. The origin of transplanted cells were further confirmed by positive immunolabeling with anti-human nuclei. Our results suggest that enriching the neuroprogenitor population by magnetic sorting prevents tumor formation and is a prerequisite before cell replacement therapy for Parkinson's disease.
Human embryonic stem cells offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent downstream manipulation. Prior to considering therapeutic applications, it is crucial that the cells are surveyed at a genetic and proteomic level during the extensive propagation, expansion and differentiation. Hence, a set of characterization tests to measure stem cell stability and identity--genomic, epigenomic and mitochondrial markers, as well as functional measures of utility, need to be developed. Thus, we outline a plan of standard assays that can be afforded by multiple laboratories to unambiguously test the quality of human embryonic stem cells. In this manuscript, we describe a comprehensive characterization of ReliCell hES1, the only human embryonic stem cell line reported from the Indian subcontinent. Our study employs gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, mitochondrial DNA sequencing, microRNA analysis, immunophenotyping and teratoma formation, in addition to demonstrating its capacity to propagate under feeder-free conditions.
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