Larry Low-Tone Ho

Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes

hMSCs derived from bone marrow are useful as a species‐specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN‐16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP‐hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP‐hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP‐hMSCs showed no signs of neoplastic transformation as examined by soft‐agar anchorage‐independent growth and NOD‐SCID mouse tumorigenicity assays. KP‐hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34dimCD38+CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP‐hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.

Significant Variation of the Elevated Nitric Oxide Levels in Aqueous Humor from Patients with Different Types of Glaucoma

Though several studies have shown that the biochemical function of nitric oxide (NO) in the eye might play an important role in the regulation of intraocular pressure (IOP), local control of ocular blood flow and loss of retinal ganglion cells by apoptosis, it is unclear whether the role of NO is similar in the pathogenesis of different kinds of glaucoma: primary open-angle glaucoma (POAG), chronic closed-angle glaucoma (CCAG) and neovascular glaucoma (NVG). To further explore this issue, we measured the concentrations of NO in aqueous humor and plasma samples from patients with POAG (n = 31), CCAG (n = 76), NVG (n = 8) and cataract (n = 30). All of the NVG patients suffered from severe proliferative diabetic retinopathy, while other patients were free of any other systemic disease. The NO levels in both aqueous humor and plasma samples were assessed by chemiluminescence assay. We found that the NO levels in aqueous humor samples were greatly varied in patients with POAG (36.2 ± 3.3 µM), CCAG (47.7 ± 3.4 µM) and NVG (65.8 ± 5.4 µM), and all of them were significantly higher than in cataract patients (27.0 ± 2.9 µM; p < 0.05). Except NVG patients whose NO levels in plasma samples were highest (24.1 ± 3.5 µM) among all groups, the plasma NO levels were not significantly different between the other glaucoma patients and the cataract patients. We therefore concluded that significant variation of the elevated NO levels in aqueous humor samples from the patients with different types of glaucoma may reflect their differences in the pathogenesis.

Lineage Differentiation‐Associated Loss of Adenoviral Susceptibility and Coxsackie‐Adenovirus Receptor Expression in Human Mesenchymal Stem Cells

Previous reports debated the effects of differentiation on adenoviral vector (AdV) transduction efficiency and Cox‐sackie‐adenovirus receptor (CAR) expression. This prompted us to investigate the efficiency of AdV transduc‐tion and CAR expression in human mesenchymal stem cells (hMSCs) and their differentiated progeny. Current results revealed high efficiency (>90%) of AdV transduction and a consistent level of CAR expression in hMSCs by the use of AdV carrying the enhanced green fluorescent protein reporter gene. Competition of CAR with blocking monoclonal antibody RmcB resulted in a reduction in transduction efficiency, indicating the CAR involvement in transduction of hMSCs. The cells were then induced to differentiate into bone, fat, or neural cells, and results demonstrated that the differentiation was accompanied with a consistent decline in AdV transduction and a decrement in CAR expression. Cells were infected with AdV and then induced into differentiation, and results demonstrated that transduced cells preserved differentiation potentials and still had transgene expression in a subpopulation of cells for 4 weeks and even in tested lineage‐specific differentiation. According to the present investigation, undifferentiated hMSCs can serve as a gene‐delivering system, and gene transfer into hMSCs before differentiation can resolve the difficulties in transduction of their differentiated progeny.

Apoptosis of Human Retina and Retinal Pigment Cells Induced by Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) retinitis is the most common ocular opportunistic infection in immunocompromised patients and AIDS. It often leads to blindness if left untreated. The question as to how HCMV infection causes retinal pathogenesis and visual destruction in AIDS patients remains unresolved. To answer the question, by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, we detected the significant signals of apoptotic cells at the same sites in the HCMV-infected retina of AIDS patients as compared to AIDS patients without HCMV retinitis. In vitro study also revealed apoptosis induced by HCMV infection in human retinal pigment epithelium cells, mediated by activation of caspase 3 and poly(ADP-ribose) polymerase pathway. These results strongly suggest the fundamental role of HCMV-induced apoptosis in mediating cell death in infected human retina and retinal pigment epithelium cells to make severe visual impairment.

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor β1 (TGF-β1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-β1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.