Expression of Neurodevelopmental Markers by Cultured Porcine Neural Precursor Cells
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《干细胞学杂志》
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@choc.org
ABSTRACT
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 . A major limitation of neural transplantation, however, has been the difficulty of extending these results to adult rodents and large mammals , including humans . Recent studies suggest that this challenge may be surmountable through the use of cultured neural stem or progenitor cells instead of solid tissue grafts .
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) . 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) . 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 , rat , dog , pig , and human . In addition, we have previously shown that NPCs can be cultured from human CNS tissue after relatively prolonged postmortem intervals . 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 . The expanded porcine neural precursors have been used as donor cells and are capable of engrafting into the mammalian CNS after transplantation . 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
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 .
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.
Immunocytochemistry
Further analysis of marker expression by porcine CNS precursors was carried out using immunocytochemical (ICC) techniques. Pig forebrain and brainstem/cerebellum cultures appeared to exhibit equivalent staining patterns for the markers examined, and therefore the combined ICC data are presented here (Fig. 2). These studies showed the expression of doublecortin (DCX), glial fibrillary acidic protein (GFAP), and Sox2, and also demonstrated the presence of neural cell adhesion molecule (NCAM), polysialic acid (PSA)–NCAM, synapsin I, ?-III tubulin, and vimentin. Other proteins detected included the membrane channel aquaporin 4 (AQP4) and the proliferation marker Ki-67. All these markers have been identified in previous studies of human CNS progenitor cells . Table 3 lists all the gene products investigated, grouped by techniques used, as well as their cellular functions. As with the RT-PCR results, however, there was no clear signal to indicate expression of nestin with any of the three different antibodies tried.
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 ?-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.
RT-PCR
As an initial step toward characterization of these cells, total RNA from adherent forebrain-derived porcine precursors was examined by RT-PCR for the expression of established neurodevelopmental markers. Findings from the pig were compared with results obtained from brain-derived hNPCs. The pig cells were clearly positive for DCX, GFAP, Hes1, and Sox2, whereas the signal for nestin was very faint (Fig. 3). In contrast, hNPCs were clearly positive for expression of all five of these genes.
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 ?-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.
Gene Microarray
In an effort to increase the number of potential targets for additional marker studies, total RNA from porcine forebrain precursors was analyzed using an available microarray designed for human samples. The resulting data from the pig were then compared with that from hNPCs. With respect to expression data as a whole, the degree of variance in reported expression levels was considerably larger between the two species than seen for comparisons of human samples to each other (data not shown). In addition, the proportion of transcripts detected in human, but not pig, samples was large. Conversely, the proportion of genes reported as expressed in pig, but not human, was small (Fig. 2H).
A number of specific genes were then chosen, based on positive expression in hNPCs , 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.
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.
Flow Cytometry
Porcine forebrain precursors were also examined by flow cytometry. This study confirmed expression of the neural adhesion molecule NCAM (CD56) on the surface of these cells, as compared with IgG control (Fig. 5). Other cell surface markers known to be highly positive in hNPCs but showing no staining in porcine forebrain precursors included CD29, CD44, CD81, CD90, CD133, and CD184.
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.
Novel Markers Expressed by hNPCs
Of the markers found to be positively expressed by hNPCs, some have not been previously reported for these cells. These markers include FABP7, Hes1, nogoA, Pbx1, and SDF1. The remainder have been mentioned in previous studies .
DISCUSSION
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 .
ACKNOWLEDGMENTS
Klassen H, Lund RD. Retinal transplants can drive a pupillary reflex in host rat brains. Proc Natl Acad Sci U S A 1987;84:6958–6960.
Coffey PJ, Lund RD, Rawlins JN. Retinal transplant-mediated learning in a conditioned suppression task in rats. Proc Natl Acad Sci U S A 1989;86:7248–7249.
Klassen H, Lund RD. Retinal graft-mediated pupillary responses in rats: restoration of a reflex function in the mature mammalian brain. J Neurosci 1991;10:578–587.
Bragadottir R, Narfstrom K. Lens sparing pars plana vitrectomy and retinal transplantation in cats. Vet Ophthalmol 2003;6:135–139.
Radtke ND, Aramant RB, Seiler MJ et al. Vision change after sheet transplant of fetal retina with retinal pigment epithelium to a patient with retinitis pigmentosa. Arch Ophthalmol 2004;122:1159–1165.
Klassen HJ, Ng TF, Kurimoto Y et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Invest Ophthalmol Vis Sci 2004;45:4167–4173.
Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707–1710.
Richards LJ, Kilpatrick TJ, Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci U S A 1992;89:8591–8595.
Seaberg RM, van der Kooy D. Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci 2003;26:125–131.
Sah DW, Ray J, Gage FH. Regulation of voltage- and ligand-gated currents in rat hippocampal progenitor cells in vitro. J Neurobiol 1997;32:95–110.
Milward EA, Lundberg CG, Ge B et al. Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain. J Neurosci Res 1997;50:862–871.
Smith PM, Blakemore WF. Porcine neural progenitors require commitment to the oligodendrocyte lineage prior to transplantation in order to achieve significant remyelination of demyelinated lesions in the adult CNS. Eur J Neurosci 2000;12:2414–2424.
Svendsen CN, Clarke DJ, Rosser AE et al. Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system. Exp Neurol 1996;137:376–388.
Palmer TD, Schwartz PH, Taupin P et al. Progenitor cells from human brain after death. Nature 2001;411:42–43.
Schwartz PH, Bryant P, Fuja T et al. Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res 2003;74:838–851.
Klassen H, Ziaeian B, Kirov I et al. Isolation of retinal progenitor cells from postmortem human tissue with comparison to autologous brain progenitors. J Neurosci Res 2004;77:334–343.
Isacson O, Deacon TW. Specific axon guidance factors persist in the adult brain as demonstrated by pig neuroblasts transplanted to the rat. Neuro-science 1996;7:827–837.
Armstrong RJ, Harrower TP, Hurelbrink CB et al. Porcine neural xeno-grafts in the immunocompetent rat: immune response following grafting of expanded neural precursor cells. Neuroscience 2001;106:201–216.
Uchida K, Okano H, Hayashi T et al. Grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons. J Neurosci Res 2003;72:61–69.
Armstrong RJ, Hurelbrink CB, Tyers P et al. The potential for circuit reconstruction by expanded neural precursor cells explored through porcine xenografts in a rat model of Parkinson’s disease. Exp Neurol 2002;175:98–111.
Armstrong RJ, Tyers P, Jain M et al. Transplantation of expanded neural precursor cells from the developing pig ventral mesencephalon in a rat model of Parkinson’s disease. Exp Brain Res 2003;151:204–217.
Dahlstrand J, Collins VP, Lendahl U. Expression of the class VI intermediate filament nestin in human central nervous system tumors. Cancer Res 1992;52:5334–5341.
Mokry J, Nemecek S. Cerebral angiogenesis shows nestin expression in endothelial cells. Gen Physiol Biophys 1999;18(suppl 1):25–29.
Alliot F, Rutin J, Leenen PJ et al. Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin. J Neurosci Res 1999;58:367–378.
Cai J, Wu Y, Mirua T et al. Properties of a fetal multipotent neural stem cell (NEP cell). Dev Biol 2002;251:221–240.
Popperl H, Bienz M, Studer M et al. Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell 1995;81:1031–1042.
Mann RS, Affolter M. Hox proteins meet more partners. Curr Opin Genet Dev 1998;8:423–429.
Roberts VJ, van Dijk MA, Murre C. Localization of Pbx1 transcripts in developing rat embryos. Mech Dev 1995;51:193–198.
Redmond L, Hockfield S, Morabito MA. The divergent homeobox gene PBX1 is expressed in the postnatal subventricular zone and interneurons of the olfactory bulb. J Neurosci 1996;16:2972–2982.
Tsai RY, McKay RD. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev 2002;16:2991–3003.
Nico B, Paola Nicchia G, Frigeri A et al. Altered blood-brain barrier development in dystrophic MDX mice. Neuroscience 2004;125:921–935.
Badaut J, Brunet JF, Grollimund L et al. Aquaporin 1 and aquaporin 4 expression in human brain after subarachnoid hemorrhage and in peritumoral tissue. Acta Neurochir 2003;86(suppl):495–498.
Moody DE, Zou Z, McIntyre L. Cross-species hybridisation of pig RNA to human nylon microarrays. BMC Genomics 2002;3:27.
Jones DG, Redpath CM. Regeneration in the central nervous system: pharmacological intervention, xenotransplantation, and stem cell transplantation. Clin Anat 1998;11:263–270.(Philip H. Schwartza,c, Hu)
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@choc.org
ABSTRACT
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 . A major limitation of neural transplantation, however, has been the difficulty of extending these results to adult rodents and large mammals , including humans . Recent studies suggest that this challenge may be surmountable through the use of cultured neural stem or progenitor cells instead of solid tissue grafts .
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) . 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) . 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 , rat , dog , pig , and human . In addition, we have previously shown that NPCs can be cultured from human CNS tissue after relatively prolonged postmortem intervals . 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 . The expanded porcine neural precursors have been used as donor cells and are capable of engrafting into the mammalian CNS after transplantation . 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
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 .
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.
Immunocytochemistry
Further analysis of marker expression by porcine CNS precursors was carried out using immunocytochemical (ICC) techniques. Pig forebrain and brainstem/cerebellum cultures appeared to exhibit equivalent staining patterns for the markers examined, and therefore the combined ICC data are presented here (Fig. 2). These studies showed the expression of doublecortin (DCX), glial fibrillary acidic protein (GFAP), and Sox2, and also demonstrated the presence of neural cell adhesion molecule (NCAM), polysialic acid (PSA)–NCAM, synapsin I, ?-III tubulin, and vimentin. Other proteins detected included the membrane channel aquaporin 4 (AQP4) and the proliferation marker Ki-67. All these markers have been identified in previous studies of human CNS progenitor cells . Table 3 lists all the gene products investigated, grouped by techniques used, as well as their cellular functions. As with the RT-PCR results, however, there was no clear signal to indicate expression of nestin with any of the three different antibodies tried.
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 ?-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.
RT-PCR
As an initial step toward characterization of these cells, total RNA from adherent forebrain-derived porcine precursors was examined by RT-PCR for the expression of established neurodevelopmental markers. Findings from the pig were compared with results obtained from brain-derived hNPCs. The pig cells were clearly positive for DCX, GFAP, Hes1, and Sox2, whereas the signal for nestin was very faint (Fig. 3). In contrast, hNPCs were clearly positive for expression of all five of these genes.
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 ?-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.
Gene Microarray
In an effort to increase the number of potential targets for additional marker studies, total RNA from porcine forebrain precursors was analyzed using an available microarray designed for human samples. The resulting data from the pig were then compared with that from hNPCs. With respect to expression data as a whole, the degree of variance in reported expression levels was considerably larger between the two species than seen for comparisons of human samples to each other (data not shown). In addition, the proportion of transcripts detected in human, but not pig, samples was large. Conversely, the proportion of genes reported as expressed in pig, but not human, was small (Fig. 2H).
A number of specific genes were then chosen, based on positive expression in hNPCs , 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.
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.
Flow Cytometry
Porcine forebrain precursors were also examined by flow cytometry. This study confirmed expression of the neural adhesion molecule NCAM (CD56) on the surface of these cells, as compared with IgG control (Fig. 5). Other cell surface markers known to be highly positive in hNPCs but showing no staining in porcine forebrain precursors included CD29, CD44, CD81, CD90, CD133, and CD184.
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.
Novel Markers Expressed by hNPCs
Of the markers found to be positively expressed by hNPCs, some have not been previously reported for these cells. These markers include FABP7, Hes1, nogoA, Pbx1, and SDF1. The remainder have been mentioned in previous studies .
DISCUSSION
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 .
ACKNOWLEDGMENTS
Klassen H, Lund RD. Retinal transplants can drive a pupillary reflex in host rat brains. Proc Natl Acad Sci U S A 1987;84:6958–6960.
Coffey PJ, Lund RD, Rawlins JN. Retinal transplant-mediated learning in a conditioned suppression task in rats. Proc Natl Acad Sci U S A 1989;86:7248–7249.
Klassen H, Lund RD. Retinal graft-mediated pupillary responses in rats: restoration of a reflex function in the mature mammalian brain. J Neurosci 1991;10:578–587.
Bragadottir R, Narfstrom K. Lens sparing pars plana vitrectomy and retinal transplantation in cats. Vet Ophthalmol 2003;6:135–139.
Radtke ND, Aramant RB, Seiler MJ et al. Vision change after sheet transplant of fetal retina with retinal pigment epithelium to a patient with retinitis pigmentosa. Arch Ophthalmol 2004;122:1159–1165.
Klassen HJ, Ng TF, Kurimoto Y et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Invest Ophthalmol Vis Sci 2004;45:4167–4173.
Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707–1710.
Richards LJ, Kilpatrick TJ, Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci U S A 1992;89:8591–8595.
Seaberg RM, van der Kooy D. Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci 2003;26:125–131.
Sah DW, Ray J, Gage FH. Regulation of voltage- and ligand-gated currents in rat hippocampal progenitor cells in vitro. J Neurobiol 1997;32:95–110.
Milward EA, Lundberg CG, Ge B et al. Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain. J Neurosci Res 1997;50:862–871.
Smith PM, Blakemore WF. Porcine neural progenitors require commitment to the oligodendrocyte lineage prior to transplantation in order to achieve significant remyelination of demyelinated lesions in the adult CNS. Eur J Neurosci 2000;12:2414–2424.
Svendsen CN, Clarke DJ, Rosser AE et al. Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system. Exp Neurol 1996;137:376–388.
Palmer TD, Schwartz PH, Taupin P et al. Progenitor cells from human brain after death. Nature 2001;411:42–43.
Schwartz PH, Bryant P, Fuja T et al. Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res 2003;74:838–851.
Klassen H, Ziaeian B, Kirov I et al. Isolation of retinal progenitor cells from postmortem human tissue with comparison to autologous brain progenitors. J Neurosci Res 2004;77:334–343.
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