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Expression of Neurodevelopmental Markers by Cultured Porcine Neural Precursor Cells

^a National Human Neural Stem Cell Resource and
^b Stem Cell Research, Children’s Hospital of Orange County Research Institute, Orange, California, USA;
^c Developmental Biology Center, School of Biological Sciences, and
^d Department of Ophthalmology, College of Medicine, University of California, Irvine, California, USA;
^e Schepens Eye Research Institute and Department of Ophthalmology, Harvard University School of Medicine, Boston, Massachusetts, USA

Key Words. Neural stem cells • Progenitor cells • Brain • Nestin • Sox2 • Pig • Human

Correspondence: Henry Klassen, M.D., Ph.D., Director, Stem Cell Research, Children’s Hospital of Orange County, Research Institute, 455 South Main Street, Orange, California 92868-3874, USA. Telephone: 714-516-4280; Fax: 714-289-4531; e-mail: hklassen{at}choc.org

	ABSTRACT

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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, ß-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.

	INTRODUCTION

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The devastating consequences of central nervous system (CNS) diseases have motivated the search for effective treatments, with much attention devoted to the possibility of neuronal replacement. One strategy is neural transplantation, in which developmentally immature donor tissue is grafted to selected sites within the host CNS. This approach has been shown to restore a number of visual functions in rodent models using grafts of fetal neural tissue [1, 2]. A major limitation of neural transplantation, however, has been the difficulty of extending these results to adult rodents [3] and large mammals [4], including humans [5]. Recent studies suggest that this challenge may be surmountable through the use of cultured neural stem or progenitor cells instead of solid tissue grafts [6].

It is now well established that developmentally immature precursor cells can be isolated from the CNS of developing rodents and propagated for extended periods in culture using mitogens such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) [7, 8]. When shown to meet specific criteria for self-renewal and multipotency, these cells are referred to as neural stem cells (NSCs) or multipotent neural progenitor cells (NPCs) [9]. In cases in which the cultured population has been less well defined, or appears to be particularly heterogeneous, use of the broader term neural precursors is often preferred. For example, when growing these cells from large mammals, cellular heterogeneity and senescence are characteristic findings. Furthermore, detailed characterization in terms of gene expression and marker studies is limited by the availability of reagents that work reliably in the particular species under investigation. Neural progenitors or precursors have now been derived from the brains of a number of mammalian species, including mouse [7, 8], rat [10], dog [11], pig [12], and human [13]. In addition, we have previously shown that NPCs can be cultured from human CNS tissue after relatively prolonged postmortem intervals [14–16]. The degree to which these various cultured neural populations have been characterized, however, varies considerably.

A number of previous studies have demonstrated that NPCs can be derived from the pig brain and propagated in culture using EGF and bFGF as mitogens [12, 17–19]. The expanded porcine neural precursors have been used as donor cells and are capable of engrafting into the mammalian CNS after transplantation [17–21]. These cells therefore provide a large animal comparison for human NPCs (hNPCs) and have been considered as a potential alternative source for donor material, yet relatively little is known about these cells. Porcine neural precursors reportedly express markers of neural lineage under differentiation conditions; however, apart from nestin, little has been reported with respect to marker expression under baseline proliferation conditions. Major disadvantages of working with these novel cells are the paucity of pig-specific antibodies, together with the lack of a comprehensive database for the porcine genome. Here we apply reagents derived for use in other mammalian species to the further characterization of porcine neural precursors and identify a number of markers also expressed in hNPC cultures. Evidence of similarities between human and porcine NPCs is relevant when interpreting the results of pig transplantation studies in the context of potential clinical trials in humans.

	MATERIALS AND METHODS

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Donor Animals and Human Subjects
Porcine fetuses were obtained off-site from a pregnant sow placed under general anesthesia and terminated prior to waking, according to a protocol approved by the Institutional Animal Care and Use Committee. Informed consent for the donation of human brain tissue was acquired prior to tissue acquisition, under the auspices of the protocol for the National Human Neural Stem Cell Resource as approved by the Children’s Hospital of Orange County Institutional Review Board. All tissues, both porcine and human, were acquired in compliance with National Institutes of Health and institutional guidelines.

Cells
The techniques used for cell isolation of the hNPCs used in this study were described previously [14–16]. The isolation of porcine cells followed a similar protocol, albeit with fetal pigs collected at 60 days of gestational age (total gestational period ~114 days). Briefly, the cranium was opened, brains removed, and the forebrain separated from the cerebellum and brainstem. Neural tissue was enzymatically digested, and the resulting cell suspension was washed repeatedly and cultured at high density in fibronectin-coated flasks containing Dulbecco’s modified Eagle’s medium/F-12 with high glucose (Irvine Scientific, Santa Ana, CA, http://www.irvinesci.com), L-glutamine (200 mM), BIT9500 (10% by volume; StemCell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com), EGF (20 ng/ml), bFGF (40 ng/ml), platelet-derived growth factor–AB (20 ng/ml), and antibiotics. Fetal bovine serum (10% by volume) was included overnight to promote adherence, and the media was completely changed the next day. Subsequently, cells were fed by 50% media exchange every 2–3 days and passaged at confluence using Cell Dissociation Buffer (Gibco, Grand Island, NY, http://www.invitrogen.com) and gentle trituration.

Candidate Markers and Genes
The markers and gene products examined in this study (Table 1) were suggested either by previous work with mammalian CNS progenitor cells from species other than pig or, in some cases, by analysis of human gene microarray data.

Flow Cytometry
Samples were handled in the manner described previously for flow cytometry, using the same antibodies [15]. Briefly, cells were harvested enzymatically as a single cell suspension, incubated with labeled-primary or primary and labeled-secondary antibodies, and analyzed on a FACS Vantage cell sorter with an Enterprise 488-nm argon laser (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). The isotype control used was mouse IgG, clone MOPC. Up to 30,000 events per sample were collected for analysis.

	RESULTS

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Proliferative cultures were obtained from fetal pig brain, including both forebrain and pooled brainstem/cerebellum samples. These cultures could be grown as adherent monolayers (Fig. 1) or as suspended spheres. Both adherent cells and spheres exhibited morphological characteristics consistent with other mammalian neural progenitor cultures, including analogous cells from humans [13–15].

View larger version (88K): [in this window] [in a new window]   Figure 1. Porcine neural precursor cells in culture. (A): Cultures derived from forebrain and plated on fibronectin grew as an adherent monolayer under proliferation conditions. Inset shows enlarged view of field from same image. (B): Equivalent cultures derived from cerebellum/brainstem, grown under identical conditions, also formed an adherent monolayer. Inset shows low-power view of neurosphere from separate culture of the same cells. All images are phase-contrast; scale bar = 50 µm.

View larger version (37K): [in this window] [in a new window]   Figure 2. Immunocytochemical analysis of porcine CNS precursors. (A): Widespread nuclear staining for Sox2 (red), subset of cells with cytoskeletal staining for GFAP (blue). (B): Widespread nuclear staining for Ki-67 (red). (C): Surface staining of numerous small rounded profiles for PSA-NCAM (red), subset of broader profiles staining for GFAP (green). (D): Widespread cytoskeletal staining of broad profiles for vimentin (green), subset of smaller profiles with fine processes staining for synapsin I (red). (E): Subset of small bi- or tripolar profiles stain for DCX (red). (F): Profiles with long, thin processes staining for ß-III tubulin (red). (G): Subset of small, rounded profiles with punctate surface staining for aquaporin-4 (green). (H): Scatter plot of human microarray data from pig and human brain stem cell (pBSC, hBSC) cultures showing relative proportion of genes "present" in human only (purple), pig only (blue), or both (yellow). Scale bars = 50 µm. Abbreviations: CNS, central nervous system; DCX, doublecortin; GFAP, glial fibrillary acidic protein; PSA-NCAM, polysialic acid–neural cell adhesion molecule.

View larger version (86K): [in this window] [in a new window]   Figure 3. Neurodevelopmental markers by RT-PCR: pig versus human. Primers designed for detection of human transcripts (Table 1) were used to evaluate proliferative porcine and human forebrain cultures. For each gene, four alternating lanes from left to right contained porcine product (Pig+), porcine negative control (Pig–), human product (Hum+), and human negative control (Hum–). Porcine precursors showed expression of DCX, GFAP, Hes1, and Sox2, yet there was little signal for nestin. Human progenitors clearly expressed all five genes. Positive control was ß-actin. Products are shown together with a 100-bp ladder at the left of the gel and predicted product sizes are in the right margin. Abbreviations: DCX, doublecortin; GFAP, glial fibrillary acidic protein; RT-PCR, reverse transcription–polymerase chain reaction.

A number of specific genes were then chosen, based on positive expression in hNPCs [15, 16], and the expression data from the pig were examined with respect to these loci. Of this subset, genes reported as positive in the pig included Hes1, Sox2, and vimentin, consistent with the RT-PCR and ICC studies reported above, as well as the new targets nogoA (RTN4) and stromal cell–derived factor 1 (SDF1). Genes not detected in the pig by human microarray, despite having been demonstrated by the initial RT-PCR and ICC studies, included DCX and GFAP. Also not detected by microarray were transcripts for the commonly used markers CD133, Ki-67, nestin, and nucleostemin. Closer examination of the data revealed an additional subset of genes showing some oligomers as positively or marginally detected, yet others absent. Examples include CXCR4 (CD184, fusin), cyclin D2, fatty acid binding protein 7 (FABP7), and Pbx1. A number of these candidate genes were selected for further analysis using RT-PCR with human primer sequences.

Additional RT-PCR
The second set of RT-PCR experiments was directed toward validating the microarray data. This work revealed positive expression by pig forebrain precursors of cyclin D2, nucleostemin, nogoA, Pbx1, and vimentin (Fig. 4). No signal was detected for CD133, FABP7, Ki-67, or SDF1. Positive expression of all nine of these genes was verified in human forebrain NPCs.

View larger version (83K): [in this window] [in a new window]   Figure 4. RT-PCR evaluation of candidate genes: pig versus human. Human primers (Table 1) were used to evaluate proliferative porcine and human forebrain cultures. For each gene, four alternating lanes from left to right contained porcine product, porcine negative control, human product, and human negative control, all labeled as in Figure 3. Porcine precursors showed expression of cyclin D2, Pbx1, nogoA, vimentin, and nucleostemin, but no product was detected for Ki-67, SDF1, CD133, or FABP7. Human progenitors clearly expressed all nine genes. A 100-bp ladder is at the left of the gel and predicted product sizes are in the right margin. Abbreviations: FABP7, fatty acid binding protein 7; RT-PCR, reverse transcription–polymerase chain reaction; SDF1, stromal cell–derived factor 1.

View larger version (13K): [in this window] [in a new window]   Figure 5. Flow cytometric analysis of porcine forebrain precursors. (A): Baseline fluorescence of isotype control. (B): Positive expression of NCAM by pig forebrain precursors. Horizontal axis = relative fluorescence; vertical axis = counts. Abbreviation: NCAM, neural cell adhesion molecule.

	DISCUSSION

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Previous work on the characterization of porcine neural precursor cells has been limited in terms of marker studies, in part because of a shortage of pig-specific genetic data and reagents. In particular, previous molecular characterization of these cells in the undifferentiated state has been limited to evaluation of nestin expression. The current study shows that detection of nestin in pig cells can itself be problematic when using a number of readily available reagents, but that a range of additional markers can be detected and that these are consistent with immature CNS precursors, including cytoskeletal proteins, surface markers, cell cycle–related proteins, and nuclear transcription factors. This study, therefore, increases our understanding of neural precursors in large animals and provides data bearing on the validity and limitations of available techniques for further characterizing these cells.

The intermediate filament protein nestin is highly expressed during development of the vertebrate CNS and has been considered a useful, if imperfect, marker of NSCs. For instance, nestin is not a surface marker and can be expressed by a number of normal and abnormal CNS cell types [22–24]. Previous studies have reported nestin expression in proliferative precursor populations cultured from the porcine brain [19, 20]. These studies used ICC techniques to demonstrate the presence of nestin-associated epitopes. In contrast, the current study was unable to demonstrate substantial nestin expression, either by immunocytochemistry or RT-PCR. Because nestin expression is a hallmark of vertebrate CNS development, this result can be reasonably attributed to inadequate detection, presumably as a result of the particular antihuman reagents used here, despite the fact that several different antibody preparations were tried. Whereas many proteins can be detected across a range of species by a given antibody, nestin is more specific, generally requiring the use of different monoclonal antibodies for detection in rodents and humans. Similarly, detection of nestin transcripts by RT-PCR benefits from species-specific primers. Because of the noted tendency toward species-related restriction in the specificity of nestin epitopes and primers, the discrepancy in the nestin findings between the current study and previous reports is most likely related to the use of different antibodies. In this case, the development of pig-specific antibodies and primers would provide the optimal solution to reliable nestin detection in porcine cells and tissues.

In addition to nestin, a number of other markers have been associated with neural stem and progenitor cells. The present study shows expression of the transcription factors Sox2 and Hes1, the surface marker PSA-NCAM, and the intermediate filament vimentin, all of which have been reported as expressed by CNS precursor cells, or subsets thereof [25, 15]. In addition, the present study provides evidence from RT-PCR for expression of Pbx1, a homologue of the Drosophila extradenticle (exd) homeodomain protein [26]. Pbx proteins are known to act as cofactors for other homeodomain transcription factors [27]. Pbx1 has not been previously identified in cultured neural precursor populations; however, it is known to be expressed at high levels in the developing nervous system [28]. Studies in the rat have shown that Pbx1 is expressed by proliferating cells of the subventricular zone, as well as their neuronal progeny, but not by glia [29]. In the context of previous in vivo work, the results of the current study showing Pbx1 expression in cultured neural precursors from the pig and human forebrain suggest a significant role for this gene in mammalian neurogenesis.

Another feature of immature precursor cell cultures is proliferative activity, reflected in the expression of genes involved in the cell cycle and its regulation. In this context, the nucleolar marker nucleostemin [30] was expressed by both pig and human neural precursors, as was cyclin D2. In contrast to the findings with nestin, the commonly used proliferation marker Ki-67, readily detected in human cells by ICC, microarray, and RT-PCR, was identified in porcine cells, albeit by immunocytochemistry alone. No signal was obtained from porcine samples using the RT-PCR primers or the human gene microarray. Again, this seemingly paradoxical finding presumably relates to differences in the human and porcine coding sequences for this gene, together with conservation of the immunologically recognized epitope of the expressed protein.

In terms of phenotypic potential, porcine neural precursor cultures included subsets of cells that expressed markers of either neuronal or glial lineages. The neural cell adhesion molecule NCAM (CD56) is widely expressed throughout the developing and mature CNS. DCX, ß-III tubulin, and synapsin I are expressed by neuronal precursors, whereas GFAP is associated with astroglia, as well as neural precursors. The expression of this latter intermediate filament protein by cultured porcine neural precursors has been previously reported [20] and is replicated here. AQP4 is a water channel found in the plasma membrane, particularly of glial vascular processes (endfeet) and may play a role in the formation of the blood–brain barrier [31, 32].

Cross-species hybridization on microarrays has been reported previously, including work with pig RNA on human arrays [33]. Cross-species comparisons are known to be associated with increased variability of the expression data, as was the case here. Some of this variability may reflect actual differences in relative gene expression between species; however, a substantial artifactual component is likely as well. Similarly, the large number of porcine transcripts undetected using the human array might be anticipated as a result of hybridization failures secondary to species-specific differences in sequence. Whereas this large body of negative data are inherently difficult to interpret, the subset of genes reported as positively expressed by both human and porcine cells represents a source of candidate genes with a high likelihood of verification upon further analysis. Indeed, the cross-species microarray analysis performed here led to the presumptive identification of cyclin D2, nogoA (RTN4), and Pbx1 expression by porcine neural progenitors, all of which were subsequently confirmed by RT-PCR (Fig. 5).

An additional consideration with respect to differences found in the data from porcine and human cells is that the cell populations studied are not entirely equivalent. In the present study, samples were taken at different gestational ages and different cerebral locations in pigs and humans. In this respect, differences in the structure and developmental time-course of the brain in these two species would appear to make sample nonequivalence unavoidable. This fact therefore makes the similarity of findings between the two species seen here all the more striking, while also reaffirming that negative marker expression in the pig might be real in some instances because of species differences or nonequivalence of cells. Additional cross-species work will be necessary to clarify these issues.

	CONCLUSION

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The present study expands considerably the number of markers known to be expressed by cultured porcine neural precursors. Of particular interest are nuclear transcription factors, both because of their highly conserved sequences, as well as their prominent role in phenotypic plasticity and lineage specification. The expression of Hes1, Pbx1, and Sox2 by forebrain precursors of both pig and human argues for an important role for these genes in the neural development of large mammals. The markers examined here were chosen based on positive expression by human forebrain progenitors and, with that in mind, the results are quite similar between the two species. Given this degree of similarity, it would seem probable that the frequent failure to detect porcine markers using human-specific primers, oligomers, and antibodies reflects specificity issues more than biological differences in expression. The development of pig-specific reagents would be helpful and is likely to increase as interest grows in the use of the pig as a large animal model in a variety of experimental paradigms, including stem cell transplantation [34].

	ACKNOWLEDGMENTS

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The authors would like to thank Dr. Tasneem Zahir for assistance with this project. The work was supported by the CHOC Foundation, Guilds, and Padrinos (H.K., P.H.S.), the Hoag Foundation (H.K.), the United Mitochondrial Research Foundation (P.H.S.), Richard D. and Gail Siegal (M.J.Y.), the Minda de Gunzburg Center for Retinal Transplantation (M.J.Y.), the NINDS (NS044060, H.K.), and the NEI (EY09595, M.J.Y.).

DISCLOSURES
The authors indicate no potential conflicts of interest.

	REFERENCES

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