Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line.

The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile(148)Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.

Clinical-grade human neural stem cells promote reparative neovascularization in mouse models of hindlimb ischemia.

OBJECTIVE: CTX0E03 (CTX) is a clinical-grade human neural stem cell (hNSC) line that promotes angiogenesis and neurogenesis in a preclinical model of stroke and is now under clinical development for stroke disability. We evaluated the therapeutic activity of intramuscular CTX hNSC implantation in murine models of hindlimb ischemia for potential translation to clinical studies in critical limb ischemia. APPROACH AND RESULTS: Immunodeficient (CD-1 Fox(nu/nu)) mice acutely treated with hNSCs had overall significantly increased rates and magnitude of recovery of surface blood flow (laser Doppler), limb muscle perfusion (fluorescent microspheres, P<0.001), and capillary and small arteriole densities in the ischemic limb (fluorescence immunohistochemistry, both P<0.001) when compared with the vehicle-treated group. Hemodynamic and anatomic improvements were dose related and optimal at a minimum dose of 3×10(5) cells. Dose-dependent improvements in blood flow and increased vessel densities by hNSC administration early after ischemia were confirmed in immunocompetent CD-1 and streptozotocin-induced diabetic mice, together with marked reductions in the incidence of necrotic toes (P<0.05). Delayed administration of hNSCs, 7 days after occlusion, produced restorative effects when comparable with acute treatment of 35 days after hindlimb ischemia. Histological studies in hindlimb ischemia immunocompetent mice for the first 7 days after treatment revealed short-term hNSC survival, transient elevation of early host muscle inflammatory, and angiogenic responses and acceleration of myogenesis. CONCLUSIONS: hNSC therapy represents a promising treatment option for critical limb ischemia.

Development of pluripotent stem cells for vascular therapy.

Peripheral arterial disease (PAD) is characterized by reduced limb blood flow due to arterial obstruction. Current treatment includes surgical or endovascular procedures, the failure of which may result in amputation of the affected limb. An emerging therapeutic approach is cell therapy to enhance angiogenesis and tissue survival. Small clinical trials of adult progenitor cell therapies have generated promising results, although large randomized clinical trials using well-defined cells have not been performed. Intriguing pre-clinical studies have been performed using vascular cells derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSCs). In particular, hiPSC-derived vascular cells may be a superior approach for vascular regeneration. The regulatory roadmap to the clinic will be arduous, but achievable with further understanding of the reprogramming and differentiation processes; with meticulous attention to quality control; and perseverance.

Intracranial delivery of stem cells.

The method of delivery of stem cells is a major factor to consider in the design of clinical trials of cell therapy. Different methods of delivery will be associated with different risks to the patient, and may also be associated with different potential for benefit. Current approaches are partly informed by the routes selected for study in animal models of focal ischaemia and CNS transplantation, but there has been little work comparing the efficacy of different routes of administration. Direct intraparenchymal delivery of cells has been employed in several preliminary clinical trials, and data on the safety of this approach are reviewed.

Immortalized human fetal retinal cells retain progenitor characteristics and represent a potential source for the treatment of retinal degenerative disease.

Human fetal retinal cells have been widely advocated for the development of cellular replacement therapies in patients with retinal dystrophies and age-related macular degeneration. A major limitation, however, is the lack of an abundant and renewable source of cells to meet therapeutic demand, although theoretically this may be addressed through the use of immortalized retinal progenitor cell lines. Here, we have used the temperature-sensitive tsA58 simian virus SV40 T antigen to conditionally immortalize human retinal progenitor cells isolated from retinal tissue at 10-12 weeks of gestation. We show that immortalized human fetal retinal cells retain their progenitor cell properties over many passages, and are comparable with nonimmortalized human fetal retinal cultures from the same gestational period with regard to expression of certain retinal genes. To evaluate the capacity of these cells to integrate into the diseased retina and to screen for potential tumorigenicity, cells were grafted into neonatal hooded Lister rats and RCS dystrophic rats. Both cell lines exhibited scarce integration into the host retina and failed to express markers of mature differentiated retinal cells. Moreover, although immortalized cells showed a greater propensity to survive, the cell lines demonstrated poor long-term survival. All grafts were infiltrated with host macrophage/microglial cells throughout their duration of survival. This study demonstrates that immortalized human fetal retinal progenitor cells retain their progenitor characteristics and may therefore have therapeutic potential in strategies that demand a renewable and consistent supply of donor cells for the treatment of degenerative retinal diseases.

Stem cell treatment of ischemic brain injury.

The development of a neural stem cell (NSC) treatment for ischemic brain injury would be of great benefit. This review focuses on recent advances in knowledge and practices toward the development of a successful NSC therapy for patients who have experienced stroke. The data supporting potential mechanisms by which somatic stem cells orchestrate neuroprotection, neurogenesis and vascularization, leading to pronounced functional improvements in stroke-induced deficits are critically reviewed. A model is proposed in which the host's immune system plays a central role in stem cell-induced neural repair. In addition, the source of stem cells and the effect of immunosuppression in clinical trials of stem cell therapy are discussed. The beneficial effects of NSCs in animal models of stroke, combined with a consideration of clinical requirements, can potentially provide an effective NSC treatment for patients who have experienced stroke.

c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.

BACKGROUND: The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively. RESULTS: The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence. CONCLUSION: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

Growth kinetics and transplantation of human retinal progenitor cells.

We studied the growth kinetics of human retinal progenitor cells (hRPCs) isolated from donor tissue of different gestational ages (G.A.), determined whether hRPCs can be differentiated into mature photoreceptors and assessed their ability to integrate with degenerating host retina upon transplantation. Eyes (12-18 weeks G.A.) were obtained with IRB approval and retinas were enzymatically dissociated. Cells were expanded in vitro, counted at isolation and at each passage, and characterized using immunocytochemistry and PCR. GFP positive hRPCs were co-cultured with retinal explants from rd1 and rhodopsin -/- mice, or transplanted into B6 mice with retinal photocoagulation and rhodopsin -/- mice. Eyes were harvested for histological evaluation following transplantation. Our results show that hRPCs from 16 to 18 weeks G.A. had the longest survival in vitro and yielded the maximum number of cells, proliferating over at least 6 passages. These cells expressed the retinal stem cell markers nestin, Ki-67, PAX6 and Lhx2, and stained positively for photoreceptor markers upon differentiation with serum. Some of the GFP positive cells used for transplantation studies showed evidence of migration into the degenerative host retina and expressed rhodopsin. In conclusion, we have determined the growth kinetics of hRPCs and have shown that cells from donor tissue of 16-18 weeks G.A. exhibit the best proliferative dynamics under the specified conditions, and that hRPCs can also be differentiated along the photoreceptor lineage. Further, we have also demonstrated that following transplantation, some of these cells integrate within the host retina and differentiate to express rhodopsin, thereby supporting the potential utility of hRPC transplantation in the setting of retinal degenerative disorders.

Molecular characterization of human retinal progenitor cells.

PURPOSE: To examine the molecular profile of fetal human retinal progenitor cells (hRPCs) expanded in vitro and those grown in a co-culture system with mouse retina through the analysis of protein and gene expression and neurotransmitter-stimulated calcium dynamics. METHODS: hRPCS were isolated from human retina of 14 to 18 weeks gestational age (GA) and expanded in vitro. Immunoblot, microarray, and immunocytochemistry (ICC) assays were performed on undifferentiated hRPCs and those co-cultured with mouse retinas for 2 weeks. Cell function was assessed by using calcium imaging. RESULTS: The ICC results showed a gradual decrease in the percentages of KI67-, SOX2-, and vimentin-positive cells from passages (P) 1 to P6, whereas a sustained expression of nestin and PAX6 was observed through P6. Microarray analysis of P1 hRPCs showed the expression of early retinal developmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2, HES5, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). At P6, hRPCs continued to express VIM, KI67, NES, PAX6, SOX2, GNL3, and SIX6. On co-culture, there was a significant increase in the expression of MKI67, PAX6, SOX2, GNL3, SIX3, and RHO (rhodopsin). Calcium imaging showed a functional response to excitatory neurotransmitters. CONCLUSIONS: Fetal-derived hRPCs show molecular characteristics indicative of a retinal progenitor state up to P6 (latest passage studied). They show a progressive decrease in the expression of immature markers as they reach P6. These cells are functional, respond to excitatory neurotransmitters, and exhibit changes in expression patterns in response to co-culture with mouse retina.

Implantation of c-mycER TAM immortalized human mesencephalic-derived clonal cell lines ameliorates behavior dysfunction in a rat model of Parkinson's disease.

Human neural stem cells offer the hope that a cell therapy treatment for Parkinson's disease (PD) could be made widely available. In this study, we describe two clonal human neural cell lines, derived from two different 10-week-old fetal mesencephalic tissues and immortalized with the c-mycER(TAM) transgene. Under the growth control of 4-hydroxytamoxifen, both cell lines display stable long-term growth in culture with a normal karyotype. In vitro, these nestin-positive cells are able to differentiate into tyrosine hydroxylase (TH)-positive neurons and are multipotential. Implantation of the undifferentiated cells into the 6-OHDA substantia nigral lesioned rat model displayed sustained improvements in a number of behavioral tests compared with noncell-implanted, vehicle-injected controls over the course of 6 months. Histological analysis of the brains showed survival of the implanted cells but no evidence of differentiation into TH-positive neurons. An average increase of approximately 26% in host TH immunoreactivity in the lesioned dorsal striatum was observed in the cell-treated groups compared to controls, with no difference in loss of TH cell bodies in the lesioned substantia nigra. Further analysis of the cell lines identified a number of expressed trophic factors, providing a plausible explanation for the effects observed in vivo. The exact mechanisms by which the implanted human neural cell lines provide behavioral improvements in the PD model are not completely understood; however, these findings provide evidence that cell therapy can be a potent treatment for PD acting through a mechanism independent of dopaminergic neuronal cell replacement.

PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons.

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.

Differential development of neuronal physiological responsiveness in two human neural stem cell lines.

BACKGROUND: Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to neurodegenerative disease. Overexpression of the myc family transcription factors in human primary cells from developing cortex and mesencephalon has produced two stable multipotential NSC lines (ReNcell VM and CX) that can be continuously expanded in monolayer culture. RESULTS: In the undifferentiated state, both ReNcell VM and CX are nestin positive and have resting membrane potentials of around -60 mV but do not display any voltage-activated conductances. As initially hypothesized, using standard methods (stdD) for differentiation, both cell lines can form neurons, astrocytes and oligodendrocytes according to immunohistological characteristics. However it became clear that this was not true for electrophysiological features which designate neurons, such as the firing of action potentials. We have thus developed a new differentiation protocol, designated 'pre-aggregation differentiation' (preD) which appears to favor development of electrophysiologically functional neurons and to lead to an increase in dopaminergic neurons in the ReNcell VM line. In contrast, the protocol used had little effect on the differentiation of ReNcell CX in which dopaminergic differentiation was not observed. Moreover, after a week of differentiation with the preD protocol, 100% of ReNcell VM featured TTX-sensitive Na+-channels and fired action potentials, compared to 25% after stdD. Currents via other voltage-gated channels did not appear to depend on the differentiation protocol. ReNcell CX did not display the same electrophysiological properties as the VM line, generating voltage-dependant K+ currents but no Na+ currents or action potentials under either stdD or preD differentiation. CONCLUSION: These data demonstrate that overexpression of myc in NSCs can be used to generate electrophysiologically active neurons in culture. Development of a functional neuronal phenotype may be dependent on parameters of isolation and differentiation of the cell lines, indicating that not all human NSCs are functionally equivalent.

A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke.

Transplantation of neural stem cells into the brain is a novel approach to the treatment of chronic stroke disability. For clinical application, safety and efficacy of defined, stable cell lines produced under GMP conditions are required. To this end, a human neural stem cell line, CTX0E03, was derived from human somatic stem cells following genetic modification with a conditional immortalizing gene, c-mycER(TAM). This transgene generates a fusion protein that stimulates cell proliferation in the presence of a synthetic drug 4-hydroxy-tamoxifen (4-OHT). The cell line is clonal, expands rapidly in culture (doubling time 50-60 h) and has a normal human karyotype (46 XY). In the absence of growth factors and 4-OHT, the cells undergo growth arrest and differentiate into neurons and astrocytes. Transplantation of CTX0E03 in a rat model of stroke (MCAo) caused statistically significant improvements in both sensorimotor function and gross motor asymmetry at 6-12 weeks post-grafting. In addition, cell migration and long-term survival in vivo were not associated with significant cell proliferation. These data indicate that CTX0E03 has the appropriate biological and manufacturing characteristics necessary for development as a therapeutic cell line.

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM).

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.

Tbx5 and Tbx4 transcription factors interact with a new chicken PDZ-LIM protein in limb and heart development.

The T-domain transcription factors, Tbx5 and Tbx4, play important roles in vertebrate limb and heart development. To identify interacting and potential Tbx-regulating proteins, we performed a yeast two-hybrid screen with the C-terminal domain of Tbx5 as bait. We identified a new PDZ-LIM protein composed of one N-terminal PDZ and three C-terminal LIM domains, which we named chicken LMP-4. Among the Tbx2, 3, 4, 5 subfamily, we observed exclusive interaction with Tbx5 and Tbx4 proteins. Tbx3 nor Tbx2 can substitute for LMP-4 binding. While chicken LMP-4 associates with Tbx5 or Tbx4, it uses distinct LIM domains to bind to the individual proteins. Subcellular co-localization of LMP-4 and Tbx proteins supports the protein interaction and reveals interference of LMP-4 with Tbx protein distribution, tethering the transcription factors to the cytoskeleton. The protein-protein interaction indicates regulation of Tbx function at the level of transcription factor nuclear localization. During chicken limb and heart development, Tbx5/LMP-4 and Tbx4/LMP-4 are tightly co-expressed in a temporal and spatial manner, suggesting that they operate in the same pathway. Surprisingly, chicken LMP-4 expression domains outside those of Tbx5 in the heart led to the discovery of Tbx4 expression in the outflow tract and the right ventricle of this organ. The Tbx4-expressing cells coincide with those of the recently discovered secondary anterior heart-forming field. The discrete posterior or anterior expression domains in the heart and the exclusive fore- or hindlimb expression of Tbx5 and Tbx4, respectively, suggest common pathways in the heart and limbs. The identification of a new Tbx5/4-specific binding factor further suggests a novel mechanism for Tbx transcription factor regulation in development and disease.

Regulation of growth factor receptors by gangliosides.

Since their discovery in the 1940s, gangliosides have been associated with a number of biological processes, such as growth, differentiation, and toxin uptake. Hypotheses about regulation of these processes by gangliosides are based on indirect observations and lack a clear definition of their mechanisms within the cell. The first insights were provided when a reduction in cell proliferation in the presence of gangliosides was attributed to inhibition of the epidermal growth factor receptor (EGFR). Since that initial finding, most, if not all, growth factor receptors have been described as regulated by gangliosides. In this review, we describe the effects of gangliosides on growth factor receptors, beginning with a list of known effects of gangliosides on growth factor receptors; we then present three models based on fibroblast growth factor (FGFR), platelet-derived growth factor receptor (PDGFR), and EGFR. We focus first on ganglioside modulation of ligand binding; second, we discuss ganglioside regulation of receptor dimerization; and third, we describe a model that implicates gangliosides with receptor activation state and subcellular localization. The methodology used to develop the three models may be extended to all growth factor receptors, bearing in mind that the three models may not be mutually exclusive. We believe that gangliosides do not act independently of many well-established mechanisms of receptor regulation, such as clathrin-coated pit internalization and ubiquitination, but that gangliosides contribute to these functions and to signal transduction pathways. We hypothesize a role for the diverse structures of gangliosides in biology through the organization of the plasma membrane into lipid raft microdomains of unique ganglioside composition, which directly affect the signal duration and membrane localization of the growth factor receptor.

Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.