Publications

 

Later Passage Neural Progenitor Cells from Neonatal Brain Are More Permissive for Human Cytomegalovirus Infection.

 Pan X, Li XJ, Liu XJ, Yuan H, Li JF, Duan YL, Ye HQ, Fu YR, Qiao GH, Wu CC, Yang B, Tian XH, Hu KH, Miao LF, Chen XL, Zheng J, Rayner S, Schwartz PH, Britt WJ, Xu J, Luo MH.

 J Virol. 2013 Jul 31. [Epub ahead of print]

 Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early passage (<passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study NPC cultures derived at different gestational ages were evaluated after short (passage 3-6) and extended (passage11-20) in vitro passage for biological and virological parameters (i.e. cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by gestational age of the source tissues. However, extended passage cultures showed evidence of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and extended passage NPCs are differentiated and more permissive for HCMV infection. Later passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.

 

Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.

Stover AE, Brick DJ, Nethercott HE, Banuelos MG, Sun L, O’Dowd DK, Schwartz PH.

 J Neurosci Res. 2013 Jul 26. doi: 10.1002/jnr.23245. [Epub ahead of print]

 Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.

 

Cancer stem cells from a rare form of glioblastoma multiforme involving the neurogenic ventricular wall.

Li SC, Vu LT, Ho HW, Yin HZ, Keschrumrus V, Lu Q, Wang J, Zhang H, Ma Z, Stover A, Weiss JH, Schwartz PH, Loudon WG.

Cancer Cell Int. 2012 Sep 20;12(1):41. doi: 10.1186/1475-2867-12-41.

BACKGROUND: The cancer stem cell (CSC) hypothesis posits that deregulated neural stem cells (NSCs) form the basis of brain tumors such as glioblastoma multiforme (GBM). GBM, however, usually forms in the cerebral white matter while normal NSCs reside in subventricular and hippocampal regions. We attempted to characterize CSCs from a rare form of glioblastoma multiforme involving the neurogenic ventricular wall.

METHODS: We described isolating CSCs from a GBM involving the lateral ventricles and characterized these cells with in vitro molecular biomarker profiling, cellular behavior, ex vivo and in vivo techniques.

RESULTS:The patient’s MRI revealed a heterogeneous mass with associated edema, involving the left subventricular zone. Histological examination of the tumor established it as being a high-grade glial neoplasm, characterized by polygonal and fusiform cells with marked nuclear atypia, amphophilic cytoplasm, prominent nucleoli, frequent mitotic figures, irregular zones of necrosis and vascular hyperplasia. Recurrence of the tumor occurred shortly after the surgical resection. CD133-positive cells, isolated from the tumor, expressed stem cell markers including nestin, CD133, Ki67, Sox2, EFNB1, EFNB2, EFNB3, Cav-1, Musashi, Nucleostemin, Notch 2, Notch 4, and Pax6. Biomarkers expressed in differentiated cells included Cathepsin L, Cathepsin B, Mucin18, Mucin24, c-Myc, NSE, and TIMP1. Expression of unique cancer-related transcripts in these CD133-positive cells, such as caveolin-1 and -2, do not appear to have been previously reported in the literature. Ex vivo organotypic brain slice co-culture showed that the CD133+ cells behaved like tumor cells. The CD133-positive cells also induced tumor formation when they were stereotactically transplanted into the brains of the immune-deficient NOD/SCID mice.

CONCLUSIONS:This brain tumor involving the neurogenic lateral ventricular wall was comprised of tumor-forming, CD133-positive cancer stem cells, which are likely the driving force for the rapid recurrence of the tumor in the patient

 

Viral mitochondria-localized inhibitor of apoptosis (UL37 exon 1 protein) does not protect human neural precursor cells from human cytomegalovirus-induced cell death.

 Hildreth RL, Bullough MD, Zhang A, Chen HL, Schwartz PH, Panchision DM, Colberg-Poley AM.

 J Gen Virol. 2012 Nov;93(Pt 11):2436-46. doi: 10.1099/vir.0.044784-0. Epub 2012 Aug 8.

Congenital human cytomegalovirus (HCMV) infection can cause severe brain abnormalities. Apoptotic HCMV-infected brain cells have been detected in a congenitally infected infant. In biologically relevant human neural precursor cells (hNPCs), cultured in physiological oxygen tensions, HCMV infection (m.o.i. of 1 or 3) induced cell death within 3 days post-infection (p.i.) and increased thereafter. Surprisingly, its known anti-apoptotic genes, including the potent UL37 exon 1 protein (pUL37x1) or viral mitochondria-localized inhibitor of apoptosis (vMIA), which protects infected human fibroblasts (HFFs) from apoptosis and from caspase-independent, mitochondrial serine protease-mediated cell death, were expressed by 2 days p.i. Consistent with this finding, an HCMV UL37x1 mutant, BADsubstitutionUL37x1 (BADsubUL37x1) induced cell death in hNPCs (m.o.i. = 1) to level which were indistinguishable from parental virus (BADwild-type)-infected hNPCs. Surprisingly, although BADsubUL37x1 is growth defective in permissive HFFs, it produced infectious progeny in hNPCs with similar kinetics and to levels comparable to BADwild-type-infected hNPCs (m.o.i. = 1). While delayed at a lower multiplicity (m.o.i. = 0.3), the BADsubUL37x1 mutant reached similar levels to revertant within 12 days, in contrast to its phenotype in HFFs. The inability of pUL37x1/vMIA to protect hNPCs from HCMV-induced cell death did not result from impaired trafficking as pUL37x1/vMIA trafficked efficiently to mitochondria in transfected hNPCs and in HCMV-infected hNPCs. These results establish that pUL37x1/vMIA, although protective in permissive HFFs, does not protect HCMV-infected hNPCs from cell death under physiologically relevant oxygen tensions. They further suggest that pUL37x1/vMIA is not essential for HCMV growth in hNPCs and has different cell type-specific roles.

 

A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus.

 

Duan Y, Miao L, Ye H, Yang C, Fu B, Schwartz PH, Rayner S, Fortunato EA, Luo MH.

 

Acta Biochim Biophys Sin (Shanghai). 2012 Jul;44(7):597-605. doi: 10.1093/abbs/gms041. Epub 2012 Jun 1.

 

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that 0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.

 

Noninvasive diagnosis of mucopolysaccharidosis via depth-resolved optical spectroscopy of the outer ear.

Mittal R, Schwartz PH, Brick DJ, Lieber CA.

Biomed Opt Express. 2011 Oct 1;2(10):2741-8. doi: 10.1364/BOE.2.002741. Epub 2011 Sep 6.

Current diagnostics for lysosomal storage disorders such as mucopolysaccharidosis (MPS) rely on evaluation of ex vivo bodily fluids, which has several shortcomings. In this study, we evaluated whether Raman spectroscopy could noninvasively diagnose MPS in a murine model. Via confocal sampling of the murine outer ear, Raman spectra were obtained at multiple depths. Partial least-squares discriminant analysis of the processed Raman spectra showed a 93% sensitivity and 91% specificity for disease. The discriminant algorithm relied on several Raman bands related to glycosaminoglycans (GAGs) that typically accumulate in MPS. These findings indicate the possibility for a new, noninvasive diagnostic tool for MPS.

 

The generation of embryoid bodies from feeder-based or feeder-free human pluripotent stem cell cultures.

Stover AE, Schwartz PH.

Methods Mol Biol. 2011;767:391-8. doi: 10.1007/978-1-61779-201-4_28.

Embryoid body (EB) formation is a traditional method of inducing differentiation of pluripotent stem cells (PSCs). It is a routine in vitro test of pluripotency as well as the first stage in many differentiation protocols targeted toward the production of a specific lineage or cellular population, as in neural differentiation (see Chapters 29 and 30). The induction of differentiation via EB formation is fairly straightforward. However, depending on the specific PSC culture conditions – substrate, feeders, medium, and eventual cell type of interest – various methods are applied in order to most routinely obtain healthy EB cultures.

 

Immunocytochemical analysis of human pluripotent stem cells.

Nethercott HE, Brick DJ, Schwartz PH.

Methods Mol Biol. 2011;767:201-20. doi: 10.1007/978-1-61779-201-4_15.

 This chapter will describe the most common immunocytochemical method utilized in the stem cell field – using fluorescently tagged secondary antibodies to detect a primary antibody that is bound to an epitope on a molecule of interest. Secondary antibodies recognize the heavy chain of the primary antibody’s isotype. Generally, these methods employ an incubation period of the sample with the primary antibody, a series of washes to remove unbound primary antibody, a secondary incubation period of the sample with the fluorescently conjugated secondary antibody, followed by washes and preparation for microscopy.

 

Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.

Stover AE, Schwartz PH.

Methods Mol Biol. 2011;767:137-46. doi: 10.1007/978-1-61779-201-4_10.

This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is strikingly different from traditional PSC culture, where the cells are co-cultured with feeder cells and in medium containing serum replacement. PSCs cultured in this new system have a different morphology than those cultured on feeder cells but retain their characteristic pluripotency. This feeder-free PSC culture system is conceptually similar to feeder-free systems that use mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM) and Matrigel substratum. Instead of MEF-CM, a very complex and undefined medium, this new system uses StemPro SFM, a chemically defined medium that permits enzymatic passaging with Accutase to disaggregate the colonies into single cells. Accutase passaging has been used in conjunction with Stempro in our hands for 20+ passages without detectable karyotypic abnormalities. We will also review techniques for adapting cultures previously grown on MEFs, routine passaging of the cells, and cryopreservation.

 

Traditional human embryonic stem cell culture.

Schwartz PH, Brick DJ, Nethercott HE, Stover AE.

Methods Mol Biol. 2011;767:107-23. doi: 10.1007/978-1-61779-201-4_8.

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures require daily attention. Once hESC cultures are established, they can, with skill and the methods described, be kept in continuous culture for many years. hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs and have evolved considerably in the years since the first hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture – they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently using the characterization methods, such as karyotyping, immunocytochemistry, gene expression analysis, and flow cytometry, provided in this manual.

 

Derivation of induced pluripotent stem cells by lentiviral transduction.

 

Nethercott HE, Brick DJ, Schwartz PH.

 

Methods Mol Biol. 2011;767:67-85. doi: 10.1007/978-1-61779-201-4_6.

 

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.

 

The stem cell laboratory: design, equipment, and oversight.

 Wesselschmidt RL, Schwartz PH.

 Methods Mol Biol. 2011;767:3-13. doi: 10.1007/978-1-61779-201-4_1.

 This chapter describes some of the major issues to be considered when setting up a laboratory for the culture of human pluripotent stem cells (hPSCs). The process of establishing a hPSC laboratory can be divided into two equally important parts. One is completely administrative and includes developing protocols, seeking approval, and establishing reporting processes and documentation. The other part of establishing a hPSC laboratory involves the physical plant and includes design, equipment and personnel. Proper planning of laboratory operations and proper design of the physical layout of the stem cell laboratory so that meets the scope of planned operations is a major undertaking, but the time spent upfront will pay long-term returns in operational efficiency and effectiveness. A well-planned, organized, and properly equipped laboratory supports research activities by increasing efficiency and reducing lost time and wasted resources.

 

Xenotransplantation of human neural progenitor cells to the subretinal space of nonimmunosuppressed pigs.

 

Warfvinge K, Schwartz PH, Kiilgaard JF, la Cour M, Young MJ, Scherfig E, Klassen H.

 

J Transplant. 2011;2011:948740. doi: 10.1155/2011/948740. Epub 2011 Jun 1.

 

To investigate the feasibility of transplanting human neural progenitor cells (hNPCs) to the retina of nonimmunosuppressed pigs, cultured hNPCs were injected into the subretinal space of 5 adult pigs after laser burns were applied to promote donor cell integration. Postoperatively, the retinal vessels appeared normal without signs of exudation, bleeding, or subretinal elevation. Eyes were harvested at 10-28 days. H&E consistently showed mild retinal vasculitis, depigmentation of the RPE, and marked mononuclear cell infiltrate in the choroid adjacent to the site of transplantation. Human-specific antibodies revealed donor cells in the subretinal space at 10-13 days and smaller numbers within the retina on days 12 and 13, with evidence suggesting a limited degree of morphological integration; however, no cells remained at 4 weeks. The strong mononuclear cell reaction and loss of donor cells indicate that modulation of host immunity is likely necessary for prolonged xenograft survival in this model.

 

Neural stem/progenitors and glioma stem-like cells have differential sensitivity to chemotherapy.

Gong X, Schwartz PH, Linskey ME, Bota DA.

Neurology. 2011 Mar 29;76(13):1126-34. doi: 10.1212/WNL.0b013e318212a89f. Epub 2011 Feb 23.

OBJECTIVES:
New data suggest that glioma stem-like cells (GSCs) and neural stem/progenitor cells (NSCs) may share common origins. GSCs drive tumor proliferation and appear to be resistant to classic chemotherapy, while the effects of chemotherapy on NSCs are not well studied. As the role of NSCs in learning and memory is increasingly recognized, we need to identify drugs that reduce neurotoxicity but are still effective against glial tumors.

METHODS:
We treated 3 human NSC cultures and multiple low- and high-grade GSC cultures with the commonly used agents temozolomide (TMZ) and cisplatin (CIS), and with 2 newer, promising drugs: the proteasome inhibitor bortezomib (BTZ) and the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (ERL). We measured cell survival, proliferation, cell death induction, and drug resistance markers.

RESULTS:
TMZ decreased NSC viability, while minimally affecting GSCs. TMZ induced NSC death, which was partially compensated for by increased proliferation. CIS had similar effects. The NSC’s sensitivity to TMZ and CIS correlated with low expression of the multidrug resistance gene ABCG2, but not of MGMT or MSH1/MLH2. BTZ caused an 80%decrease in GSCs, while minimally affecting NSCs. GSCs had lower proteasome levels and activity after BTZ treatment. ERL treatment also decreased GSC numbers, but not NSC viability, which correlated with low EGFR expression in NSCs compared to GSCs.

CONCLUSIONS:
Newer chemotherapy agents ERL and BTZ are effective against GSCs yet produce minimal effects on NSCs, while the older drugs TMZ and CIS are more toxic for NSCs than for GSCs. The identification and testing of more selective drugs is clearly warranted.

 

Human cytomegalovirus infection causes premature and abnormal differentiation of human neural progenitor cells.

Luo MH, Hannemann H, Kulkarni AS, Schwartz PH, O’Dowd JM, Fortunato EA.

J Virol. 2010 Apr;84(7):3528-41. doi: 10.1128/JVI.02161-09. Epub 2010 Jan 13.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, largely manifested as central nervous system (CNS) disorders. The principal site of manifestations in the mouse model is the fetal brain’s neural progenitor cell (NPC)-rich subventricular zone. Our previous human NPC studies found these cells to be fully permissive for HCMV and a useful in vitro model system. In continuing work, we observed that under culture conditions favoring maintenance of multipotency, infection caused NPCs to quickly and abnormally differentiate. This phenotypic change required active viral transcription. Whole-genome expression analysis found rapid downregulation of genes that maintain multipotency and establish NPCs’ neural identity. Quantitative PCR, Western blot, and immunofluorescence assays confirmed that the mRNA and protein levels of four hallmark NPC proteins (nestin, doublecortin, sex-determining homeobox 2, and glial fibrillary acidic protein) were decreased by HCMV infection. The decreases required active viral replication and were due, at least in part, to proteasomal degradation. Our results suggest that HCMV infection causes in utero CNS defects by inducing both premature and abnormal differentiation of NPCs.

 

A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding.

 

Gopalakrishnan S, Van Emburgh BO, Shan J, Su Z, Fields CR, Vieweg J, Hamazaki T, Schwartz PH, Terada N, Robertson KD.

 

Mol Cancer Res. 2009 Oct;7(10):1622-34. doi: 10.1158/1541-7786.MCR-09-0018. Epub 2009 Oct 13.

 

DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.

 

Two factor reprogramming of human neural stem cells into pluripotency.

Hester ME, Song S, Miranda CJ, Eagle A, Schwartz PH, Kaspar BK.

PLoS One. 2009 Sep 18;4(9):e7044. doi: 10.1371/journal.pone.0007044.

BACKGROUND:
Reprogramming human somatic cells to pluripotency represents a valuable resource for the development of in vitro based models for human disease and holds tremendous potential for deriving patient-specific pluripotent stem cells. Recently, mouse neural stem cells (NSCs) have been shown capable of reprogramming into a pluripotent state by forced expression of Oct3/4 and Klf4; however it has been unknown whether this same strategy could apply to human NSCs, which would result in more relevant pluripotent stem cells for modeling human disease.

METHODOLOGY AND PRINCIPAL FINDINGS:
Here, we show that OCT3/4 and KLF4 are indeed sufficient to induce pluripotency from human NSCs within a two week time frame and are molecularly indistinguishable from human ES cells. Furthermore, human NSC-derived pluripotent stem cells can differentiate into all three germ lineages both in vitro and in vivo.

CONCLUSIONS/SIGNIFICANCE:
We propose that human NSCs represent an attractive source of cells for producing human iPS cells since they only require two factors, obviating the need for c-MYC, for induction into pluripotency. Thus, in vitro human disease models could be generated from iPS cells derived from human NSCs.

 

Treatment reduces or stabilizes brain imaging abnormalities in patients with MPS I and II.

Wang RY, Cambray-Forker EJ, Ohanian K, Karlin DS, Covault KK, Schwartz PH, Abdenur JE.

Mol Genet Metab. 2009 Dec;98(4):406-11. doi: 10.1016/j.ymgme.2009.07.015. Epub 2009 Aug 5.

BACKGROUND:
The mucopolysaccharidoses (MPSs) are a family of lysosomal storage disorders caused by impaired glycosaminoglycan degradation. Characteristic brain imaging abnormalities are seen in MPS patients. This study aims to determine the effects of hematopoietic stem cell transplantation (HSCT) and/or intravenous enzyme replacement therapy (ERT) on these abnormalities.

METHODS:
A retrospective chart and brain imaging study review was conducted of MPS types I and II patients with brain magnetic resonance imaging (MRI) performed at, and following, initiation of treatment. White matter abnormalities, dilated perivascular spaces, corpus callosal abnormalities, and ventriculomegaly were scored by three independent neuroradiologists blinded to cognitive status, date of treatment initiation, and type(s) of treatment.

RESULTS:
Five patients were identified: three patients with MPS I and two with MPS II. Duration of follow-up ranged from 13 to 51 months. One patient had severe MPS I (genotype W402X/35del12) and received ERT followed by HSCT. The remaining patients had ERT only. The other two MPS I patients were cognitively normal siblings (genotype P533R/P533R) with an intermediate phenotype. One MPS II patient had moderate cognitive impairment without regression (genotype 979insAGCA); the other (genotype R8X) had normal cognition. There was very little inter-observer variation in MRI scoring. The greatest abnormalities for each patient were found at initial MRI. All patients, including the ERT-only patients, demonstrated improved or unchanged MRI scores following treatment. Severity of white matter abnormalities or dilated perivascular spaces did not correlate with cognitive impairment; as such, extensive pre-treatment MRI abnormalities were noted in the older, cognitively normal MPS I sibling. In comparison, his younger sibling had only mild MRI abnormalities at the same age, after receiving 4 years of ERT.

CONCLUSIONS:
This study represents one of the first to document the CNS effects of ERT in MPS patients utilizing serial brain MR imaging studies, and raises several important observations. Brain MRI abnormalities typically become more pronounced with age; initiation of ERT or HSCT reversed or stabilized this trend in the MPS patients studied. In addition, earlier initiation of treatment resulted in decreased severity of imaging abnormalities. Possible mechanisms for these observations include improved cerebrospinal fluid dynamics, reduced central nervous system glycosaminoglycan storage via efflux through the blood-brain barrier (BBB), repair of damaged BBB, reduction in CNS inflammation, or ERT permeability through the BBB.

 

Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.

Pistollato F, Chen HL, Rood BR, Zhang HZ, D’Avella D, Denaro L, Gardiman M, te Kronnie G, Schwartz PH, Favaro E, Indraccolo S, Basso G, Panchision DM.

Stem Cells. 2009 Jan;27(1):7-17. doi: 10.1634/stemcells.2008-0402.

Hypoxia commonly occurs in solid tumors of the central nervous system (CNS) and often interferes with therapies designed to stop their growth. We found that pediatric high-grade glioma (HGG)-derived precursors showed greater expansion under lower oxygen tension, typical of solid tumors, than normal CNS precursors. Hypoxia inhibited p53 activation and subsequent astroglial differentiation of HGG precursors. Surprisingly, although HGG precursors generated endogenous bone morphogenetic protein (BMP) signaling that promoted mitotic arrest under high oxygen tension, this signaling was actively repressed by hypoxia. An acute increase in oxygen tension led to Smad activation within 30 minutes, even in the absence of exogenous BMP treatment. Treatment with BMPs further promoted astroglial differentiation or death of HGG precursors under high oxygen tension, but this effect was inhibited under hypoxic conditions. Silencing of hypoxia-inducible factor 1alpha (HIF1alpha) led to Smad activation even under hypoxic conditions, indicating that HIF1alpha is required for BMP repression. Conversely, BMP activation at high oxygen tension led to reciprocal degradation of HIF1alpha; this BMP-induced degradation was inhibited in low oxygen. These results show a novel, mutually antagonistic interaction of hypoxia-response and neural differentiation signals in HGG proliferation, and suggest differences between normal and HGG precursors that may be exploited for pediatric brain cancer therapy.

 

Isolation of progenitor cells from GFP-transgenic pigs and transplantation to the retina of allorecipients.

Klassen H, Warfvinge K, Schwartz PH, Kiilgaard JF, Shamie N, Jiang C, Samuel M, Scherfig E, Prather RS, Young MJ.

Cloning Stem Cells. 2008 Sep;10(3):391-402. doi: 10.1089/clo.2008.0010.

Work in rodents has demonstrated that progenitor transplantation can achieve limited photoreceptor replacement in the mammalian retina; however, replication of these findings on a clinically relevant scale requires a large animal model. To evaluate the ability of porcine retinal progenitor cells to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower in conjunction with photoreceptor markers and glial fibrillary acid protein (GFAP), thus suggesting downregulation of GFP during differentiation. Following transplantation, GFP expression allowed histological visualization of integrated cells and extension of fine processes to adjacent plexiform layers. GFP expression in subretinal grafts was high in cells expressing vimentin and lower in cells expressing photoreceptor markers, again consistent with possible downregulation during differentiation. Cells survived transplantation to the injured retina of allorecipients at all time points examined (up to 10 weeks) in the absence of exogenous immune suppression without indications of rejection. These findings demonstrate the feasibility of allogeneic progenitor transplantation in a large mammal and the utility of the pig in ocular regeneration studies.

 

Regulatory networks define phenotypic classes of human stem cell lines.

Müller FJ, Laurent LC, Kostka D, Ulitsky I, Williams R, Lu C, Park IH, Rao MS, Shamir R, Schwartz PH, Schmidt NO, Loring JF.

Nature. 2008 Sep 18;455(7211):401-5. doi: 10.1038/nature07213. Epub 2008 Aug 24.

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells. We report here the creation and analysis of a database of global gene expression profiles (which we call the ‘stem cell matrix’) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of approximately 150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein-protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.

 

Neonatal neural progenitor cells and their neuronal and glial cell derivatives are fully permissive for human cytomegalovirus infection.

Luo MH, Schwartz PH, Fortunato EA.

J Virol. 2008 Oct;82(20):9994-10007. doi: 10.1128/JVI.00943-08. Epub 2008 Aug 6.

Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMV’s interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral isolates, Towne (laboratory adapted) and TR (a clinical strain), at a multiplicity of infection of 3. NPCs were fully permissive for both strains, expressing the full range of viral antigens (Ags) and producing relatively large numbers of infectious virions. NPCs infected with TR showed delayed development of cytopathic effects (CPE) and replication centers and shed less virus. This pattern of delay for TR infections held true for all cell types tested. Differentiation of NPCs was carried out for 21 days to obtain either astroglia (>95% GFAP(+)) or a 1:5 mixed neuron/astroglia population (beta-tubulin III(+)/GFAP(+)). We found that both of these differentiated populations were fully permissive for HCMV infection and produced substantial numbers of infectious virions. Utilizing a difference in plating efficiencies, we were able to enrich the neuron population to approximately 80% beta-tubulin III(+) cells. These beta-tubulin III(+)-enriched populations remained fully permissive for infection but were very slow to develop CPE. These infected enriched neurons survived longer than either NPCs or astroglia, and a small proportion were alive until at least 14 days postinfection. These surviving cells were all beta-tubulin III(+) and showed viral Ag expression. Surprisingly, some cells still exhibited extended processes, similar to mock-infected neurons. Our findings strongly suggest neurons as reservoirs for HCMV within the developing brain.

 

Training the next generation of pluripotent stem cell researchers.

Schwartz PH.

J Transl Med. 2008 Jul 23;6:40. doi: 10.1186/1479-5876-6-40.

Human pluripotent stem cells (PSCs) have the unique properties of being able to proliferate indefinitely in their undifferentiated state and of being able to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. PSCs in culture have a specific morphology and they express characteristic surface antigens and nuclear transcription factors; thus, PSC culture is very specific and requires a core skill set for successful propagation of these unique cells. Specialized PSC training courses have been extremely valuable in seeding the scientific community with researchers that possess this skill set.

 

Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.

Qiu J, Ai L, Ramachandran C, Yao B, Gopalakrishnan S, Fields CR, Delmas AL, Dyer LM, Melnick SJ, Yachnis AT, Schwartz PH, Fine HA, Brown KD, Robertson KD.

Lab Invest. 2008 Sep;88(9):910-25. doi: 10.1038/labinvest.2008.66. Epub 2008 Jul 7.

DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.

 

Differentiation of Neural Lineage Cells from Human Pluripotent Stem Cells

Schwartz, P.H., Brick, D.J., Stover, A.E., Loring, J.F., and Mueller, F.J.

Methods. 2008 Jun;45(2):142-58. doi: 10.1016/j.ymeth.2008.03.007. Epub 2008 May 29.

Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: 1) starting material, 2) induction of loss of pluripotency, 3) neural induction, 4) neural maintenance and expansion, and 5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

Stem Cell Therapies for the Lysosomal Storage Diseases – the Quintessential Neurodegenerative Diseases

Schwartz, P.H. and Brick, D.J.

Curr Stem Cell Res Ther. 2008 May;3(2):88-98.

As a novel neurotherapeutic strategy, stem cell transplantation has received considerable attention. However, little focus of this attention has been devoted to the probabilities of success of stem cell therapies for specific neurological disorders. Given the complexities of the cellular organization of the nervous system and the manner in which it is assembled during development, it seems unlikely that a cellular replacement strategy will succeed for any but the simplest of neurological disorders in the near future. A general strategy for stem cell transplantation to prevent or minimize neurological disorders is much more likely to succeed. The lysosomal storage diseases represent the quintessential neurodegenerative diseases for which preventative stem cell transplantation will both likely succeed and set the stage for therapeutic approaches to other neurodegenerative diseases.

Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes

Pistollato F, Chen HL, Schwartz, P.H., Basso G and Panchision DM

Molecular and Cellular Neuroscience 35:424-435 (2007).

Human neural precursor proliferation and potency is limited by senescence and loss of oligodendrocyte potential.We found that in vitro expansion of human postnatal brain CD133+ nestin+ precursors is enhanced at 5% oxygen, while raising oxygen tension to 20% depletes precursors and promotes astrocyte differentiation even in the presence of mitogens. Higher cell densities yielded more astrocytes regardless of oxygen tension. This was reversed by noggin at 5%, but not 20%, oxygen due to a novel repressive effect of low oxygen on bone morphogenetic protein (BMP) signaling. When induced to differentiate by mitogen withdrawal, 5% oxygen-expanded precursors generated 17-fold more oligodendrocytes than cells expanded in 20% oxygen. When precursors were expanded at 5% oxygen and then differentiated at 20% oxygen, oligodendrocyte maturation was further enhanced 2.5- fold. These results indicate that dynamic control of oxygen tension regulates different steps in fate and maturation and may be crucial for treating neurodegenerative diseases.

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease

Lee, J.P., Lee, P.J., Takahashi, H., Jeyakumar, M., Clark, D., Clarke, J., Tong, G., Wenger, D., Platt, F.M., Seyfried, T.M., Schwartz, P., Lipton, S.A., and Snyder, E.Y.

Nature Medicine 13:439-447 (2007).

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain b-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (b-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.

Neural precursors isolated from the developing cat brain show retinal integration following transplantation to the retina of the dystrophic cat

Klassen H, Schwartz, P.H., Ziaeian B, Nethercott H, Young MJ, Bragodottir R, Tullis GE, Warfvinge K and Narfstrom K

Veterinary Ophthalmology 10:245-253 (2007).

The cat has served as an important nonrodent research model for neurophysiology and retinal degenerative disease processes, yet very little is known about feline neural precursor cells. To culture these cells and evaluate marker expression, brains were dissected from 45-day-old fetuses, enzymatically dissociated, and grown in the presence of EGF, bFGF and PDGF. Expanded cells widely expressed nestin, Sox2, Ki-67, fusin (CXCR4) and vimentin, while subpopulations expressed A2B5, GFAP, or β-III tubulin. Precursors prelabeled with BrdU and/or transduced with a recombinant lentivirus that expresses GFP were transplanted subretinally in five dystrophic Abyssinian cats. Two to 4 weeks following surgery, histology showed survival of grafted cells in three of the animals. Labeled cells were found in the neuroretina and RPE layer, as well as in the vitreous and the vicinity of Bruch’s membrane. There was no evidence of an immunologic response in any of the eyes. Neural precursor cells can therefore be cultured from the developing cat brain and survive as allografts for up to 4 weeks without immune suppression. The feasibility of deriving and transplanting feline neural precursor cells, combined with the availability of the dystrophic Abyssinian cat, provide a new feline model system for the study of retinal repair.

An approach to the ethical donation of human embryos for harvest of stem cells

Schwartz PH, Rae, SB.

Reprod. Biomed. Online 2006 Jun;12(6):771-775.

This paper considers embryo grading within a given infertility treatment and suggests an ethical approach to embryo donation for embryonic stem cell harvest. It is concluded that ethical considerations regarding human embryos do not necessarily preclude the use of certain embryos for biomedical research or transplantation. The argument is based on the following rationale: all embryos are not physiologically equal, some low-grade embryos will never be chosen for implantation, cells from low-grade embryos may be donated for transplantation or research, and embryonic stem cells can be harvested from low-grade embryos. This argument bears special importance at this time as embryos created by IVF are still the only source of embryonic stem cells, given the current controversy surrounding published studies of human somatic cell nuclear transfer.

Regulation of human neural precursor cells by laminin and integrins

Flanagan LA, Rebaza LM, Derzic S, Schwartz PH, Monuki ES.

J Neurosci Res. 2006 Apr;83(5):845-56.

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.

Mammalian stem cells

Terskikh AV, Bryant PJ, Schwartz PH.

Pediatr Res. 2006 Apr;59(4 Pt 2):13R-20R.

Stem cells are quickly coming into focus of much biomedical research eventually aiming at the therapeutic applications for various disorders and trauma. It is important, however, to keep in mind the difference between the embryonic stem cells, somatic stem cells and somatic precursor cells when considering potential clinical applications. Here we provide the review of the current status of stem cell field and discuss the potential of therapeutic applications for blood and Immune system disorders, multiple sclerosis, hypoxic-ischemic brain injury and brain tumors. For the complimentary information about various stem cells and their properties we recommend consulting the National Institutes of Health stem cell resources (http://stemcells.nih.gov/info/basics).

The potential of stem cell therapies for neurological diseases

Schwartz PH.

Expert Rev Neurother. 2006 Feb;6(2):153-61.

As a novel neurotherapeutic strategy, stem cell transplantation has received considerable attention, yet little of this attention has been devoted to the probabilities of success of stem cell therapies for specific neurological disorders. Given the complexities of the cellular organization of the nervous system and the manner in which it is assembled during development, it is unlikely that a cellular replacement strategy will succeed for any but the simplest of neurological disorders in the near future. A general strategy for stem cell transplantation to prevent or minimize neurological disorders is much more likely to succeed. Two broad categories of neurological disease, inherited metabolic disorders and invasive brain tumors, are among the most likely candidates.

Neuropathology of fragile X-associated tremor/ataxia syndrome (FXTAS)

Greco CM, Berman RF, Martin RM, Tassone F, Schwartz PH, Chang A, Trapp BD, Iwahashi C, Brunberg J, Grigsby J, Hessl D, Becker EJ, Papazian J, Leehey MA, Hagerman RJ, Hagerman PJ.

Brain. 2006 Jan;129(Pt 1):243-55.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects carriers, principally males, of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Clinical features of FXTAS include progressive intention tremor and gait ataxia, accompanied by characteristic white matter abnormalities on MRI. The neuropathological hallmark of FXTAS is an intranuclear inclusion, present in both neurons and astrocytes throughout the CNS. Prior to the current work, the nature of the associations between inclusion loads and molecular measures (e.g. CGG repeat) was not defined. Post-mortem brain and spinal cord tissue has been examined for gross and microscopic pathology in a series of 11 FXTAS cases (males, age 67-87 years at the time of death). Quantitative counts of inclusion numbers were performed in various brain regions in both neurons and astrocytes. Inclusion counts were compared with specific molecular (CGG repeat, FMR1 mRNA level) and clinical (age of onset, age of death) parameters. In the current series, the three most prominent neuropathological characteristics are (i) significant cerebral and cerebellar white matter disease, (ii) associated astrocytic pathology with dramatically enlarged inclusion-bearing astrocytes prominent in cerebral white matter and (iii) the presence of intranuclear inclusions in both brain and spinal cord. The pattern of white matter pathology is distinct from that associated with hypertensive vascular disease and other diseases of white matter. Spongiosis was present in the middle cerebellar peduncles in seven of the eight cases in which those tissues were available for study. There is inclusion formation in cranial nerve nucleus XII and in autonomic neurons of the spinal cord. The most striking finding is the highly significant association between the number of CGG repeats and the numbers of intranuclear inclusions in both neurons and astrocytes, indicating that the CGG repeat is a powerful predictor of neurological involvement in males, both clinically (age of death) and neuropathologically (number of inclusions).

Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells

Arocena DG, Iwahashi CK, Won N, Beilina A, Ludwig AL, Tassone F, Schwartz PH, Hagerman PJ.

Hum Mol Genet. 2005 Dec 1;14(23):3661-71.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects some adult carriers of pre-mutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. FXTAS is thought to be caused by a toxic ‘gain-of-function’ of the expanded CGG-repeat FMR1 mRNA, which is found in the neuronal and astrocytic intranuclear inclusions associated with the disorder. Using a reporter construct with a FMR1 5′ untranslated region harboring an expanded (premutation) CGG repeat, we have demonstrated that intranuclear inclusions can be formed in both primary neural progenitor cells and established neural cell lines. As with the inclusions found in post-mortem tissue, the inclusions induced by the expanded CGG repeat are alphaB-crystallin-positive; however, inclusions in culture are not associated with ubiquitin, indicating that incorporation of ubiquitinated proteins is a later event in the disease process. The absence of ubiquitinated proteins also argues against a model in which inclusion formation is due to a failure of the proteasomal degradative machinery. The presence of the expanded CGG repeat, as RNA, results in reduced cell viability as well as the disruption of the normal architecture of lamin A/C within the nucleus. This last observation, and the findings that lamin A/C is present in both the inclusions of FXTAS patients and the inclusions in cell culture, suggests that lamin A/C dysregulation may be a component of the pathogenesis of FXTAS; in particular, the Charcot-Marie-Tooth-type neuropathy associated with FXTAS may represent a functional laminopathy.

Expression of neurodevelopmental markers by cultured porcine neural precursor cells

Schwartz PH, Nethercott H, Kirov II, Ziaeian B, Young MJ, Klassen H.

Stem Cells. 2005 Oct;23(9):1286-94.

Despite the increasing importance of the pig as a large animal model, little is known about porcine neural precursor cells. To evaluate the markers expressed by these cells, brains were dissected from 60-day fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor. Porcine neural precursors could be grown as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis using reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, microarrays, and flow cytometry, and results compared with data from analogous human forebrain progenitor cells. Cultured porcine neural precursors widely expressed neural cell adhesion molecule (NCAM), polysialic acid (PSA)-NCAM, vimentin, Ki-67, and Sox2. Minority subpopulations of cells expressed doublecortin, beta-III tubulin, synapsin I, glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4) consistent with increased lineage restriction. A human microarray detected porcine transcripts for nogoA (RTN4) and stromal cell-derived factor 1 (SDF1), possibly cyclin D2 and Pbx1, but not CD133, Ki-67, nestin, or nucleostemin. Subsequent RT-PCR showed pig forebrain precursors to be positive for cyclin D2, nucleostemin, nogoA, Pbx1, vimentin, and a faint band for SDF1, whereas no signal was detected for CD133, fatty acid binding protein 7 (FABP7), or Ki-67. Human forebrain progenitor cells were positive for all the genes mentioned. This study shows that porcine neural precursors share many characteristics with their human counterparts and, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL.

Lab Chip. 2005 Apr;5(4):401-6.

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.

Neural progenitor cells from an adult patient with fragile X syndrome

Schwartz PH, Tassone F, Greco CM, Nethercott HE, Ziaeian B, Hagerman RJ, Hagerman PJ.

BMC Med Genet. 2005 Jan 14;6:2.

BACKGROUND: Currently, there is no adequate animal model to study the detailed molecular biochemistry of fragile X syndrome, the leading heritable form of mental impairment. In this study, we sought to establish the use of immature neural cells derived from adult tissues as a novel model of fragile X syndrome that could be used to more fully understand the pathology of this neurogenetic disease. METHODS: By modifying published methods for the harvest of neural progenitor cells from the post-mortem human brain, neural cells were successfully harvested and grown from post-mortem brain tissue of a 25-year-old adult male with fragile X syndrome, and from brain tissue of a patient with no neurological disease. RESULTS: The cultured fragile X cells displayed many of the characteristics of neural progenitor cells, including nestin and CD133 expression, as well as the biochemical hallmarks of fragile X syndrome, including CGG repeat expansion and a lack of FMRP expression. CONCLUSION: The successful production of neural cells from an individual with fragile X syndrome opens a new avenue for the scientific study of the molecular basis of this disorder, as well as an approach for studying the efficacy of new therapeutic agents.

Isolation of Retinal Progenitor Cells from Postmortem Human Tissue and Comparison with Autologous Brain Progenitors

Klassen, H., Ziaeian, B., Kirov, I.I., Young, M.J., Schwartz, P.H.

J. Neurosci. Res., 77:334-343 (2004), with cover.

The goal of the present study was threefold: to determine whether viable human retinal progenitor cells (hRPCs) could be obtained from cadaveric retinal tissue, to evaluate marker expression by these cells, and to compare hRPCs to human brain progenitor cells (hBPCs). Retinas were dissected from post-mortem premature infants, enzymatically dissociated, and grown in the presence of epidermal growth factor and basic fibroblast growth factor. The cells grew as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis by RT-PCR, immunocytochemistry, and flow cytometry. hBPCs derived from forebrain specimens from the same donors were grown and used for RT-PCR. Post-mortem human retinal specimens yielded viable cultures that grew to confluence repeatedly, although not beyond 3 months. Cultured hRPCs expressed a range of markers consistent with CNS progenitor cells, including nestin, vimentin, Sox2, Ki-67, GD2 ganglioside, and CD15 (Lewis X), as well as the tetraspanins CD9 and CD81, CD95 (Fas), and MHC class I antigens. No MHC class II expression was detected. hRPCs, but not hBPCs, expressed Dach1, Pax6, Six3, Six6, and recoverin. Minority subpopulations of hRPCs and hBPCs expressed doublecortin, β-III tubulin, and glial fibrillary acidic protein, which is consistent with increased lineage restriction in subsets of cultured cells. Viable progenitor cells can be cultured from the post-mortem retina of premature infants and exhibit a gene expression profile consistent with immature neuroepithelial cells. hRPCs can be distinguished from hBPC cultures by the expression of retinal specification genes and recoverin.

Redistribution of transcription start sites within the FMR1 promoter region with expansion of the downstream CGG-repeat element

Beilina, S., Tassone, F., Schwartz, P.H., Sahota, P., and Hagerman, P.J.

Hum. Mol. Genet. 13:543-549 (2004).

Fragile X syndrome, the most common form of mental impairment, is caused by expansion of a (CGG)n trinucleotide repeat element located in the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene. Repeat expansion is known to influence both transcription and translation; however, the mechanisms by which the CGG element exerts its effects are not known.

In the current work, we have utilized 5′-RLM-RACE to examine the influence of CGG repeat number on the utilization of transcription start sites in normal (n<55) and premutation (54<n<200) cell lines of both non-neural (lymphoblastoid) and neural (primary astrocyte) origin.

Our results demonstrate that, in both neural and non-neural cells, transcription of the FMR1 gene is initiated from several transcription start sites within a approximately 50 nt region that lies approximately 130 nt upstream of the CGG repeat element. For normal alleles, most transcripts initiate from the downstream-most start site, close to the single position identified previously. Surprisingly, as the size of the CGG repeat expands into the premutation range, initiation shifts to the upstream sites, suggesting that the CGG element may act as a downstream enhancer/modulator of transcription. The shift in start site selection for both neural and non-neural cells indicates that the effect is general. Furthermore, the correspondence between start site utilization and the degree of elevation of FMR1 mRNA suggests that a substantial fraction of the increased message in the premutation range may derive from the upstream start sites.

Asymmetric Localization of LGN but not AGS3, Two Homologs of Drosophila Pins, in Dividing Human Neural Progenitor Cells

Fuja, T.J., Schwartz, P.H., Darcy, D. and Bryant, P.J.

J. Neurosci. Res. 75:782-793 (2004), with cover.

Human neural progenitor cells (hNPCs) can be recovered from postmortem human brains and used to study the molecular basis of neurogenesis. Human NPCs are being used to investigate the molecular basis of cell fate determination during stem cell divisions, based on comparison with the Drosophila model system. Drosophila neuroblasts and sensory organ precursors undergo well-defined asymmetric cell divisions (ACD), under the control of a genetically defined set of apical and basal determinants that are localized tightly and dynamically during division.

We show by indirect immunofluorescence, confocal microscopy, and time-lapse video-microscopy that LGN and AGS3, two human homologs of the Drosophila ACD determinant Pins, have distinct patterns of localization in hNPCs. When cells are grown under conditions favoring proliferation, LGN is distributed asymmetrically in a cell cycle-dependent manner; it localizes to one side of the dividing cell and segregates into one of the daughter cells. When the cells are grown under conditions favoring differentiation, LGN accumulates in double foci similar to those containing the mitotic apparatus protein NuMA, and in a pattern shown previously for LGN and NuMA in differentiated cells. AGS3, a slightly more distant Pins homolog than LGN, does not show asymmetric localization in these cells. The progenitor cell marker nestin also localizes asymmetrically in colcemid-treated hNPCs and colocalizes with LGN. The results suggest that hNPCs undergo ACD and that similar molecular pathways may underlie these divisions in Drosophila and human cells.

Isolation and Characterization of Neural Progenitor Cells from Post-Mortem Human Cortex

Schwartz, P.H., Bryant, P.J., Fuja, T.J., Su, H., O’Dowd, D.K., and Klassen, H.K.

J. Neurosci. Res. 74:838-851 (2003), with cover.

Post-mortem human brain tissue represents a vast potential source of neural progenitor cells for use in basic research as well as therapeutic applications. Here we describe five human neural progenitor cell cultures derived from cortical tissue harvested from premature infants. Time-lapse videomicrography of the passaged cultures revealed them to be highly dynamic, with high motility and extensive, evanescent intercellular contacts. Karyotyping revealed normal chromosomal complements. Prior to differentiation, most of the cells were nestin, Sox2, vimentin, and/or GFAP positive, and a subpopulation was doublecortin positive. Multilineage potential of these cells was demonstrated after differentiation, with some subpopulations of cells expressing the neuronal markers beta-tubulin, MAP2ab, NeuN, FMRP, and Tau and others expressing the oligodendroglial marker O1. Still other cells expressed the classic glial marker glial fibrillary acidic protein (GFAP). RT-PCR confirmed nestin, SOX2, GFAP, and doublecortin expression and also showed epidermal growth factor receptor and nucleostemin expression during the expansion phase. Flow cytometry showed high levels of the neural stem cell markers CD133, CD44, CD81, CD184, CD90, and CD29. CD133 markedly decreased in high-passage, lineage-restricted cultures. Electrophysiological analysis after differentiation demonstrated that the majority of cells with neuronal morphology expressed voltage-gated sodium and potassium currents. These data suggest that post-mortem human brain tissue is an important source of neural progenitor cells that will be useful for analysis of neural differentiation and for transplantation studies.

Cell Culture: Progenitor cells from human brain after death

Palmer, T.D., Schwartz, P.H., Taupain, P., Kaspar, B., Stein, S.A., and Gage, F.H.

Nature 411:42-43 (2001), no abstract.