Rebecca S. Hamilton

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid β (Aβ) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimers disease (AD). Cdk5 phosphorylates tau at AD‐specific phospho‐epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Aβ peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Aβ1−42‐induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin‐dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting ‘normal’ Cdk5 activity.

Expression of tyrosine hydroxylase in an immortalized human fetal astrocyte cell line: In vitro characterization and engraftment into the rodent striatum

The use of primary human fetal tissue in the treatment of neurodegenerative disorders, while promising, faces several difficult technical and ethical issues. An alternative approach that would obviate these problems would be to use immortalized cell lines of human fetal central nervous system origin. An immortalized human fetal astrocyte cell line (SVG) has been established (45) and herein we describe the in vitro and in vivo characteristics of this cell line which suggest that it may be a useful vehicle for neural transplantation. The SVG cell line is vimentin, GFAP, Thy 1.1 and MHC class I positive, and negative for neurofilament and neuron specific enolase, consistent with its glial origin. To determine whether the cell line could be used as a drug delivery system, a cDNA expression vector for tyrosine hydroxylase was constructed (phTH/Neo) and stably expressed in the SVG cells for over 18 months as demonstrated by immunohistochemistry and Western blotting of the stable transfectants. HPLC analysis of the supernatant from these cells, termed SVG-TH, consistently found 4-6 pmol/ml/min of l-dopa produced with the addition of BH4 to the media. Furthermore, in cocultivation experiments with hNT neurons, PC-12 cells and primary rat fetal mesencephalic tissue, both the SVG and SVG-TH cells demonstrated neurotrophic potential, suggesting that they constituitively express factors with neuroregenerative potential. To determine the viability of these cells in vivo, SVG-TH cells were grafted into the striatum of Sprague-Dawley rats and followed over time. A panel of antibodies was used to unequivocally differentiate the engrafted cells from the host parenchyma, including antibodies to: SV40 large T antigen (expressed in the SVG-TH cells), human and rat MHC class 1, vimentin, GFAP, and tyrosine hydroxylase. While the graft was easily identified with the first week, over the course of a four week period of time the engrafted cells decreased in number. Concomittantly, rat CD4 and CD8 expression in the vicinity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of a 6-hydroxydopamine lesioned rats, rotational behavior of the rat decreased as much as 80% initially, then slowly returned to baseline over the next four weeks, parallelling graft rejection. Thus, the SVG-TH cells can induce a functional recovery in an animal model of Parkinsons disease, however as a xenograft, the SVG cells are recognized by the immune system.

Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins

For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease.

Differences among Isolates of Simian Hemorrhagic Fever (SHF) Virus

Abstract Simian hemorrhagic fever (SHF) virus is a member of the Togaviridae family which currently is unclassified to genus. We have studied the relatedness of four different SHF virus isolates obtained from infected macaque or patas monkeys. Differences were found among isolates in type and severity of disease produced in patas monkeys, cell sensitivity to infection, viral antigens, and levels of specific antibody induced in patas monkeys. Based on these criteria, the four isolates have been grouped in two categories: those producing acute infections in patas monkeys (LVR, P-180) and those producing persistent infections (P-248, P-741). The P-180 isolate induced the most severe disease in experimentally infected patas monkeys, but only occasionally were their infections fatal. Persistently infected patas monkeys were viremic over a period of years, but showed no signs or symptoms of infection. All four isolates were found to be antigenically related by use of enzyme-linked immunosorbent assay (ELISA); the P-248 isolate showing the weakest antigenic relationship. However, none of the four isolates induced cross-neutralizing antibodies in infected patas monkeys. High titers of specific IgG antibody (up to 31,250 as determined by ELISA) were induced in acutely infected patas monkeys (LVR, P-180), but antibody was barely detectable (≤50) in persistently infected patas monkeys (P-248, P-741). LVR lyrically infected USU-104 cells, patas monkey peritoneal macrophages (PMAC), and rhesus monkey PMAC. The P-180 isolate lytically infected both patas monkey PMAC and rhesus monkey PMAC, but not USU-104 cells. The isolates producing persistent infections (P-248, P-741) lytically infected only-rhesus monkey PMAC. These results show that marked differences exist among isolates of SHF virus from naturally infected animals. These differences should be useful in categorizing new isolates.

Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin.

Many studies have compared the genetic and epigenetic profiles of human induced pluripotent stem cells (hiPSCs) to human embryonic stem cells (hESCs) and yet the picture remains unclear. To address this, we derived a population of neural precursor cells (NPCs) from the H1 (WA01) hESC line and generated isogenic iPSC lines by reprogramming. The gene expression and methylation profile of three lines were compared to the parental line and intermediate NPC population. We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions. Analysis of the global methylation pattern also showed no significant difference between the two PSC populations. Both undifferentiated populations were distinctly different from the intermediate NPC population in both gene expression and methylation profiles. One point to note is that H1 is a male line and so extrapolation to female lines should be cautioned. However, these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real‐time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full‐length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage‐specific embryonic antigen‐1 of hESCs, following constant BMP‐4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa‐miR‐519c and hsa‐miR‐520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs. STEM Cells2012;30:2175–2187

Efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: Establishment of a glial cell line with a functional CD4 receptor

Adeno associated virus (AAV) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. We have investigated the potential for using AAV as a vector for gene transfer into glial cells of the human fetal nervous system. Recombinant AAV vectors expression either the reporter gene beta-galactosidase or a human CD4 receptor were able to transduce both primary glial cells of the human fetal nervous system and an SV40 immortalized human fetal glial cell line (SVG). No difference in transduction efficiency was observed between the primary cells and the cell line which in both cases was as high as 95%. Stable transfectants of the glial cell line expressing the CD4 receptor were selected. An SVG/CD4 expressing line was then established. The presence of the CD4 receptor was confirmed by immunohistochemistry, Westerm immuno-blotting and flow cytometric analysis. The CD4 receptor was shown to be functional by infection of the SVG/CD4 cell line with the human immunodeficiency virus (HIV). Upon infection, the SVG/CD4 cells produced 20-fold higher levels of the HIV intracellular core antigen P24 than the CD4 negative parental cells and in addition formed syncytia. The use of AAV vectors should prove useful in biological investigations of human glial cells and offers promise as a means of ex vivo and in vivo gene delivery.

Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells

Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAMpos and Tra-1-60neg by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments.

Non-colony type monolayer culture of human embryonic stem cells.

Regenerative medicine, relying on human embryonic stem cell (hESC) technology, opens promising new avenues for therapy of many severe diseases. However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress, growth rates, and heterogeneous cellular states under current culture conditions. In this study, we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states, precisely control growth rates, significantly increase cell production, and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple, robust, scalable, and suitable for high-throughput screening and drug discovery.