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Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres
Physiology & Behavior 92 (2007) 60 – 66
Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres Elisabet Åkesson a,d,⁎, Jing-Hua Piao a,d , Eva-Britt Samuelsson a , Lena Holmberg a , Anders Kjældgaard b , Scott Falci c , Erik Sundström a,d , Åke Seiger a,d a
Karolinska Institutet, Division of Neurodegeneration and Neuroinflammation, Department of Neurobiology, Care Sciences and Society, Novum, S-141 86 Stockholm, Sweden b Karolinska Institutet, Division of Obstetrics/Gynecology, Department of Clinical Sciences, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden c Craig Hospital, 3425 S. Clarkson St., Englewood, CO 80110, USA d Stockholms Sjukhem Foundation, Mariebergsgatan 22, Stockholm, Sweden
Abstract There is heterogeneity in neural stem and progenitor cell characteristics depending on their species and regional origin. In search for potent in vitro-expanded human neural precursor cells and cell therapy methods to repair the injured human spinal cord, the possible influence exerted by intrinsic cellular heterogeneity has to be considered. Data available on in vitro-expanded human spinal cord-derived cells are sparse and it has previously been difficult to establish long-term neurosphere cultures showing multipotentiality. In the present paper, human spinal cord-derived neurospheres were cultured in the presence of EGF, bFGF and CNTF for up to 25 passages (N350 days) in vitro. In contrast to the human first trimester subcortical forebrain, spinal cord tissue N 9.5 weeks of gestation could not serve as a source for long-term neurosphere cultures under the present conditions. After withdrawal of mitogens, cultured neurospheres (at 18 passages) gave rise to cells with neuronal, astrocytic and oligodendrocytic phenotypes in vitro. After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immunodeficient adult rats, large numbers of cells survived at least up to 6 weeks, expressing neuronal and astrocytic phenotypes. These results demonstrate that it is possible to expand and maintain multipotent human spinal cord-derived neurospheres in vitro for extended time-periods and that they have promising in vivo potential after engraftment to the injured spinal cord. © 2007 Elsevier Inc. All rights reserved. Keywords: Human; Embryonic; Spinal cord; Forebrain; Neurospheres; Culture; PCNA; Transplantation; Spinal cord injury
1. Introduction The possibility to expand human neural stem cells in vitro has increased the availability of potential donor cells for transplantation intervention after nervous system injury and disease. Multipotent human neural stem cells may be generated from embryonic stem (ES) cells [1] embryonic and fetal [2,3] as well as adult nervous system tissue [4]. However, multiple donor and host factors such as developmental stage, central nervous system (CNS) region and species-specific characteristics may in⁎ Corresponding author. Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Novum 5th floor, S-141 86 Stockholm, Sweden. Tel.: +46 8 58583907; fax: +46 8 58583880. E-mail address: [email protected] (E. Åkesson). 0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.05.056
fluence the in vitro and in vivo potential of neural stem cells [2,5–7]. This underlines the importance of using experimental models that mimic the clinical conditions for evaluation of the human donor cell and host properties as well as the transplantation procedure used. To repair the injured spinal cord it is of importance to evaluate the spinal cord-specific properties of the potential host and donor cells. With regard to the host, the adult spinal cord has been reported as non-neurogenic, not supporting neurogenesis of neural precursor cell transplants in allograft rodent models [8–10]. In vitro studies by Song and collaborators showed that astrocytes from adult rodent hippocampus promote neurogenesis while astrocytes from adult rodent spinal cord do not [7]. Furthermore, the conditions in the injured adult spinal cord were shown to be even less permissive than those of the normal spinal
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cord [9]. With regard to the donor cells, little is known about spinal cord-derived stem/progenitor cells. Few reports have focused on in vitro expansion of human spinal cord-derived neural precursor cells [10,11] and only one has previously shown stable long-term in vitro expansion of human spinal cord-derived neurospheres [10]. These neurospheres were shown to include only glial lineage-restricted precursor cells after differentiation. The aim of the present study was to expand multipotent human spinal cord-derived neurospheres for intraspinal transplantation. We here report successful long-term in vitro expansion of multipotent human spinal cord-derived neurospheres in the presence of ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). In addition, we found that human spinal cord-derived neural stem and progenitor cells survive and present both neuronal and glial phenotypes in vivo after engraftment to adult injured rodent spinal cord. 2. Methods and procedures 2.1. Human first trimester spinal cord and forebrain tissue Human first trimester subcortical forebrain (in the following text also referred to as forebrain) and spinal cord tissues (4.5– 12 weeks of gestation) were collected after elective routine abortions using gentle vacuum aspiration technique. Informed consent was obtained from the pregnant women. The collection was approved by the Human Ethics Committee of Huddinge University Hospital. The gestational age of the tissue was determined by examination of anatomical landmarks and the size of the abortion material according to the atlas of England [12]. The subcortical forebrain and spinal cord were dissected and freed of meninges with sterile technique in DMEM: F12 medium (Life Technologies) and divided into two pieces. One part was fixed, cryo-sectioned and prepared for immunohistochemical analyses (see below) to compare the presence of proliferating neural cells in the in situ first trimester subcortical forebrain and spinal cord tissues. The remaining part of the tissue was processed for neurosphere culture. In previous reports human fetal forebrain more frequently than spinal cord tissue has served as source for neurosphere culture. Therefore we compared the outcome of cultures from the two regions. 2.2. Human spinal cord- and forebrain-derived neurosphere cultures The tissue samples were mechanically dissociated into single cell suspensions by the use of a glass-Teflon homogenizer. The method we used for establishing and propagating neurospheres is a slight modification of the protocol developed by Carpenter and co-workers [2]. Cells were cultured in 75 ml non-coated flasks (Nunc) with 20 ml of cell suspension at a density of 100,000–150,000 cells/ml medium (DMEM: F12 at 1:1, Life Technologies) supplemented with 0.6% glucose, 5 mM Hepes, Heparin (2 μg/ml, Sigma), 1% N2-supplement (v/v, Life Technologies), EGF (20 ng/ml, R&D Systems), bFGF (20 ng/ml, R&D systems) and CNTF (10 ng/ml, R&D Systems) to form
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free-floating neurospheres. The cells were maintained at 37 °C in 5% CO2. Fresh medium was added twice a week. The neurospheres were passaged every 7–20 days (depending on the growth of the individual culture) by mechanical dissociation and then re-cultured in fresh medium as described above. All the neurosphere cultures were performed without antibiotics. For in vitro differentiation, the human neurospheres were mechanically dissociated by trituration and seeded on poly-Dlysine (Sigma, 0.1 mg/ml) and fibronectin-coated (0.1 mg/ml, Sigma) circular glass cover slips (13 mm in diameter) in 24-well plates (Nunc). The cells were seeded at a density of 26,000 cells/ ml DMEM: F12 supplemented with 0.6% glucose, 5 mM Hepes, 1% N2-supplement and 10% fetal calf serum, and cultured for 7 days prior to fixation and immunocytochemistry. To in vitro differentiate oligodendrocytes from human neurospheres a modified protocol developed by Zhang and co-workers (Zhang et al., 2000) was used, with mechanical dissociation and seeding in medium as above but in 0.5% fetal calf serum instead of 10% and the addition of 1 ng/ml recombinant human PDGF-AA (PeproTech Inc.). The medium was changed every 3 days during the 4 weeks of culture prior to fixation and immunocytochemistry. 2.3. Intraspinal transplantation of human spinal cord-derived neurospheres A spinal cord compression lesion was performed in 12 adult athymic female rats (HsdHan: RNU-rnu, Harlan, UK) weighing 170–200 g. The rats received intraperitoneal (i.p.) injections of atropine preoperatively (Atropine, 0.05 mg/kg, NM Pharma AB, Sweden) followed by i.p. injections of phenyl citrate, 0.22 mg/kg and fluanisone, 6.75 mg/kg (Hypnorm, Janssen Pharmaceutical) combined with midazolam (3.4 mg/kg, Dormicum, Roche). A partial laminectomy was made in the lower half of vertebrae L1 and a sagittal cut was made in the dura mater. Two drops of lidocain (20 mg/ml Xylocain, Astra Zeneca) was added to the exposed spinal cord just prior to the compression. The compression lesion was made by the use of a bulldog clamp with a closing pressure of 35 g (De Bakey, 58 mm in total length) applied vertically over the spinal cord for 30 s. The lesioned spinal cord was covered with one layer of meningeal substitute (Lyoplant, B/Braun Aesculap) prior to suture of the wound. Per-operative body temperature was controlled by the use of heating pads with a rectal thermometer (CMA/150, Carnegie Medicine). For post-surgical analgesia the animals were given buphrenorphine (Temgesic, 7 μg/kg, Reckitt & Colman) intramuscularly twice a day for 4 days. The urinary bladders were emptied manually twice a day. To prevent urinary infections the rats were given antibiotics (Borgal vet., 15 mg/kg, Hoechst) subcutaneously 4 days per week. Two weeks after the spinal cord compression lesions, the animals were re-anaesthetized and the lesioned area carefully exposed. Human neurospheres at passage four (P4), deriving from a 6.5–7 gestational week old spinal cord, 10–12 in number with a diameter ranging from 0.1 to 0.25 mm were injected through a glass-capillary into the spinal cord parenchyma close to the lesion area in 8 of the 12 lesioned cases. The remaining four animals underwent the same surgical procedure but with
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intraspinal injections of medium lacking neurospheres, serving as controls. After a post-surgical/transplantation period of 6 weeks, the rats were re-anesthetized once more and perfused through the ascending aorta with 100 ml calcium-free Tyrode's solution followed by 400 ml of 4% para-formaldehyde in phosphate buffer, pH 7.4. The dissected spinal cords were postfixed for another 90 min, rinsed and kept in 30% sucrose until they were cryostat-sectioned at 10 μm. The experimental procedure was approved by “The Experimental Animal Ethics Committee” of Southern Stockholm.
EDTA, 0.1% sodium dodecyl sulphate, 1% Triton X-100, Complete proteinase inhibitor (Roche) and 50 mM Tris–HCl (pH 7.6). Proteins were separated by electrophoresis on a 10% Tris– glycine sodium dodecyl sulphate-polyacrylamide gel, transferred to a nitrocellulose filter and incubated with primary antibodies towards PCNA (1:500, Santa Cruz Biotechnology). After detection of primary antisera with anti-rabbit immunoglobulin-horseradish peroxidase, the immunoblots were developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
2.4. Immunohistochemical analyses
3. Results
Formaldehyde-fixed human first trimester forebrain and spinal cord tissue was cryo-sectioned and treated with 5% acetic acid/95% ethanol (previously cooled to −20 °C) for 10 min. The sections were incubated with 1.5% normal goat serum for 30 min prior to incubation over-night with primary antiserum to proliferating cell nuclear antigen (PCNA, 1:10, Santa Cruz Biotechnology). The in vitro-differentiated human neurosphere cells were fixed with buffered 4% para-formaldehyde pH 7.4 for 10 min and rinsed in phosphate buffered saline (PBS). To reduce background the cells were incubated in 10% normal goat serum in PBS for 30 min prior to addition of the primary antisera to βtubulin III (1:800, Sigma), glial fibrillary acidic protein (GFAP, 1:500 DAKO), galactocerebroside C (GalC, 1:50, Chemicon) or O4 (1:50, gift from Dr. Pfeiffer, Univ. of Connecticut, CT). Spinal cord sections with or without transplants were incubated over-night with monoclonal antibodies to human nuclear protein (HNP, 1:250, Chemicon) and glial fibrillary acidic protein (GFAP, 1:20,000, Sternberger Monoclonals Inc.) as well as the following polyclonal antibodies raised against β-tubulin III (1:1,200, Berkeley Antibody Company), and Gal C (1:50, Chemicon) to analyze the cellular phenotypes of grafted human spinal cord-derived neurospheres. All primary antisera were diluted in 0.1 M PBS with 0.3% Triton X-100. After incubation with primary antisera over-night at + 4 °C, the sections/cells were rinsed in PBS for 3 × 10 min. Detection of primary antibodies was performed with secondary antibodies conjugated to Alexa Fluor® 488 (1:400, Molecular Probe) or Cy3 (1:800, Jackson Immunoresearch Laboratories, Inc.) added and incubated for 60 min followed by a final rinse in PBS for 3 × 10 min. Gal C immuno-labeling was visualized using a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (EnVision+™, Peroxidase, DAKO). Negative control slides were processed as above, but the primary antisera were omitted. Furthermore, extra slides with tissue sections including human neurons, astrocytes and oligodendrocytes were processed in parallel as positive controls. The images were viewed with a fluorescence microscope (Zeiss, Axiophot) and acquired using a CCD camera (Hamamatsu, ORCA-ER, C4742-95) and the Openlab software for Macintosh (Improvision).
3.1. Long-term culture of human subcortical forebrain- and spinal cord-derived neurospheres
2.5. Immunoblot Human first trimester spinal cord tissue, 5, 7, 9.5 or 10.5 weeks of gestation, were homogenized in lysis buffer comprising 2 mM
Cells isolated from human forebrain 4.5–12 weeks of gestation grew as free-floating neurospheres in the presence of EGF, bFGF and CNTF. The success rate of human neurosphere cultures, at P2, P5 and P10, with different gestational ages is summarized in Table 1. Forebrain-derived cultures could be established and sustained even from late first trimester stages. A majority of the cultures showed at least a five-fold increase in cell number at P10 compared to that at P0. The culture that expanded the most showed a 267-fold increase in cell number up to P10. A neurosphere culture deriving from forebrain tissue at 12 weeks of gestation was sustained up to at least P18. The forebrain neurospheres that were kept in culture the longest, up to P25, originated from a 5 week old embryo. Cells from human first trimester spinal cord formed neurospheres and increased in number, as long as the gestational age of the original tissue was ≤9.5 weeks of gestation. Neurospheres derived from 4.5–9.5 gestational week spinal cord could be expanded long-term, while donor stages beyond 9.5 weeks of gestation could not be propagated for more than four passages (Table 1). All the spinal cord-derived cultures, sustained up to P10, showed an increase in cell number compared to that at P0. Most of these expanded more than five-fold. The culture that expanded the most showed a 200-fold increase in cell number up to P10. The spinal cord-derived neurospheres Table 1 Human spinal cord- and forebrain-derived neurosphere cultures Gestational age
Passage (P)
Spinal cord n /n
%
ns/nt
%
≤8 weeks
2 5 10 2 5 10 2 5 10
42/45 27/31 11/15 6/8 3/6 2/5 2/6 0/5 0/5
93 87 73 75 50 40 33 0 0
42/44 18/29 12/24 5/6 4/6 4/6 8/12 4/11 3/10
95 62 50 83 75 75 67 36 30
8.5–9.5 weeks
N9.5 weeks
s
t
Forebrain
Summary over human spinal cord- and forebrain-derived cultures that resulted in sustained neurosphere formation at different donor stages and passage number (P). ns, number of sustained neurosphere cultures; nt, total number of neurosphere cultures.
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that were kept in culture the longest, up to P25 (N 350 days in culture), originated from a 7 week old embryo. 3.2. Cellular phenotypes in vitro and following intraspinal transplantation To study the cellular phenotypes of long-term cultured human neurospheres, dissociated cells were plated on cover slips and cultured without CNTF, EGF and bFGF. Both longterm cultured human forebrain- and spinal cord-derived neuro-
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spheres showed numerous β-tubulin III- and GFAP-immunoreactive (IR) cells. Gal C- and O4-IR cells were also observed but were sparse in number (Fig. 1A and B). After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immuno-deficient adult rat, numerous surviving HNP-IR cells were observed in five of seven grafted cases at 6 weeks after the graft injection, indicating human cell survival. One of the transplanted immunedeficient rodents was sacrificed prior to the endpoint due to infection and was not included in the study. No HNP-IR cells
Fig. 1. In vitro and in vivo cellular phenotypes of human spinal cord-derived neurosphere cells. A) After 18 passages in vitro, human neurospheres deriving from human spinal cord tissue, 7 weeks of gestation, were differentiated without mitogens but with fetal calf serum present in the culturing medium. GFAP- (red) and β-tubulin-IR cells (green) were numerous while B) Gal C- and O4-(see inset) IR cells were sparse in number. Scale bar = 20 μm. C) After intraspinal transplantation of human spinal cord-derived neurospheres numerous human cells including both neuronal and glial phenotypes survived in the compressionlesioned adult rat spinal cord. HNP-IR nuclei (green) with varying size and shape surrounded by nestin-IR (red) in the epicenter of transplant. D) Cells that had migrated approximately 400 μm from the transplantation site into host spinal cord showed HNP-IR nuclei (green) surrounded by nestin-IR cytoplasm and extensions (red). E) HNP- (green) and β-tubulin-IR cells (red) suggesting that the cell has neuronal phenotype. F) HNP- (green) and GFAP-IR cells (red) suggesting that these cells have astrocytic phenotypes.
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Fig. 2. Proliferating cells in in situ human forebrain and spinal cord at 7 and 10.5–11 weeks of gestation. PCNA-IR cells in the ventricular zone of 7 (A) and 10.5–11 gestational weeks old forebrain (B) and surrounding the immature central canal in the spinal cord at 7 weeks of gestation (C). The number of PCNA-IR cells was much lower in the spinal cord at 10.5–11 weeks of gestation (D) compared to that at 7 weeks of gestation. Bars = 100 μm. PCNA immunoblot of human spinal cord tissue 5– 10.5 weeks of gestation (E). The PCNA-IR bands had a molecular weight of approximately 36 kDa.
were observed in non-transplanted spinal cords. In grafted cases the vast majority of the observed HNP-IR cells also had a nestin-IR cytoplasm, suggesting that most of the grafted human cells were still neural precursors (Fig. 1C and D). However, in adjacent sections multiple cells were double-labeled with βtubulin III and HNP (Fig. 1E) or GFAP and HNP (Fig. 1F) suggesting that a subpopulation of the human-derived cells had differentiated into neuronal and glial phenotypes. The β-tubulin III-IR cells were frequently localized in groups closer to the implantation site, while the GFAP-IR cells were more scattered and seemed to have migrated further at this point in time. In contrast, no Gal C-IR was observed to be co-localized with any HNP-IR nuclei, suggesting that no grafted human neural precursor cells had differentiated into oligodendrocytes. 3.3. Proliferating cells in first trimester human CNS tissue In view of the decline in neurosphere formation and survival from the spinal cord with increasing gestational age, possible changes in the proliferating cell population of the source tissue were investigated. The presence of proliferating cells in situ in the human spinal cord and subcortical forebrain (5 to 11 weeks of gestation) was investigated by the use of an immunohistochemical marker for PCNA. We hypothesized that the decline in neurosphere formation coincides with the decrease of proliferative cells in the ventricular zone. In the subcortical forebrain
from which we readily could establish neurospheres at least as late as 12 weeks of gestation, a large number of PCNA-IR cells were observed at 7 and 10.5–11 weeks of gestation (Fig. 2A and B). On the other hand, in the in situ human spinal cord PCNA-IR cells which were abundant in the neuroepithelial cell layer at 7 weeks of gestation (Fig. 2C), were lacking in six and clearly reduced in two of the investigated eight cases at 10– 11 weeks of gestation (Fig. 2D), a fetal stage when neurospheres no longer could be established using the present protocol. Immunoblot confirmed the decline in PCNA-IR in the human spinal cord tissue from 5 to 10.5 weeks of gestation (Fig. 2E). 4. Discussion In the present study we demonstrate long-term in vitro cultures of human spinal cord-derived neurospheres with the potential to differentiate into neuronal, astrocytic and oligodendrocytic phenotypes. The long-term expansion of multipotent human spinal cord neural cells will allow us to compare their in vitro and in vivo potential with that of neural cells from other regions and other cellular sources in spinal cord repair models. Long-term maintenance of human spinal cord-derived multipotent neurospheres has previously not been described in detail according to our knowledge. Quinn et al. [10] succeeded with two cultures in the presence of EGF and bFGF up to at least 25 passages. However, in contrast to our results these cultures only
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gave rise to astrocytes suggesting that the culture conditions used selected for glial precursors. The question whether tripotent neural stem cells exist in vivo has been raised and Gabay and collaborators showed that clonogenic competence to generate neurons, astrocytes and oligodendrocytes was induced by FGF-2 deregulation of dorso-ventral patterning during expansion in vitro [13]. This induction of tripotency may occur as a result of the bFGF, EGF and CNTF presence, also in our human neurosphere cultures. The aim of cellular transplantation to a spinal cord lesion is to substitute for the extensive cell loss, be neuroprotection and support of remyelination and neuronal regeneration, in order to reach physiological restitution. Traumatic spinal cord injury (SCI) results in loss of multiple cell types. Cell therapy would preferentially include a cell population with both neuronal and glial potential. Whether a homogenous population of multipotent stem cells, a heterogeneous population of more restricted precursor cells, or a mixture of those cell types would be optimal for SCI repair is still under investigation. However, for clinical treatment it is necessary that these cells can be expanded in large numbers, maintained in vitro for months and that they remain stable with regard to differentiation potential. We suggest that human embryonic spinal cord neurosphere cultures may have this potential although further characterization of the cells is crucial to ensure stability. We have previously characterized human spinal cord-derived neurospheres concerning their immune-competence, of relevance for cell therapy application. The in vitro-expanded human neural precursor cells expressed high levels of MHC proteins on their cell surface but still did not elicit an immune response in co-cultures with peripheral human lymphocytes [14]. Using the present culturing method we succeeded in establishing and maintaining multipotent human spinal cord neurospheres long-term from donors ≤ 9.5 weeks of gestation. Beyond that developmental stage no spinal cord-derived neurospheres could be maintained for more than four passages. In the human forebrain cultures, on the other hand, we could expand neurospheres even from a case at 12 weeks of gestation. It is known, that neural stem cells remain along the entire neuraxis including the adult mammalian spinal cord [15,16]. However, despite that neurosphere formation has been reported from adult human brain cells [4,17,18] there is no report showing in vitro neurosphere formation from the adult human spinal cord. The lack of long-term neurosphere cultures from later first trimester spinal cord tissue in our hands is in concordance with the lack of initial neurosphere formation in second trimester human spinal cord specimens reported by Quinn et al. [10]. We hypothesized that human neurospheres are derived from proliferating neural precursor cells in the immature central nervous tissue. PCNA immunocytochemistry indicated that the number of dividing cells in the human spinal cord was lower at 10–11 weeks compared to that at 7 weeks of gestation, while still abundant in the subcortical forebrain at 10.5 weeks of gestation. This is in concordance with the observation of no long-term spinal cord-derived cultures beyond 9.5 weeks of gestation, while successful long-term cultures from late first
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trimester forebrain could be obtained. However, the fact that the number of PCNA-IR cells is approximately 1000 times larger than the number of forming neurospheres in vitro at the same embryonic stages (data not shown) suggests that it may be a subpopulation of the PCNA-IR cells that form the neurospheres. To propagate the human neurospheres we used the mitogens EGF and bFGF but also added the interleukin-6 family member CNTF in our cultures from initial seeding. Human forebrainderived neurosphere cultures were previously expanded in the presence of EGF, bFGF and human leukemia inhibitory factor (hLIF). Cultures grown with addition of hLIF (10 ng/ml) consistently showed an enhanced proliferation after 50–60 days in vitro, as well as an increased neuronal population after differentiation compared to equivalent cultures grown with EGF and bFGF alone [2]. For human forebrain-derived neurospheres, hLIF could be substituted with CNTF [2]. There are both interspecies and regional differences in the way neural precursor cells are regulated [5,19–21]. These factors need to be taken into account when attempting to standardize in vitro neural cell expansion conditions. With the aim to repair the injured spinal cord in man it is important to study conditions specifically necessary for expansion of human neural cells, and how they interact with the injured adult spinal cord after transplantation. Differentiation of rodent neural stem/ progenitor cells transplanted into the adult spinal cord was previously described as mainly restricted to the glial lineage [6,8,22–24]. Even neuronally restricted rodent precursor cells were inhibited to differentiate into neuronal phenotype when grafted into the contused adult rat spinal cord [9]. In the present study numerous human spinal cord-derived cells survived the transplantation to the lesioned immuno-deficient adult rat spinal cord with a large population being nestin-IR and subpopulations showing GFAP- and β-tubulin III-IR. Our human neural precursor cells showing tripotency in vitro were here shown to have the ability to differentiate into neurons and astrocytes but not oligodendrocytes (with Gal C as the immunohistochemical marker) after transplantation to the injured spinal cord. The differentiation of oligodendroglia from human fetal brainderived neurospheres, reported by others, has been limited and most oligodendroglia remain at the relatively immature O4+-stage, preceding the Gal C+-stage [1,25] that may explain why we did not observe any Gal C-IR human cells in vivo after transplantation. 5. Concluding remarks The present report demonstrates long-term in vitro expansion of human spinal cord-derived neurospheres including precursor cells with the potential to develop into astrocytes, neurons and oligodendrocytes. These neurosphere-derived spinal cord cells may in turn survive, migrate and express phenotypic proteins for neurons and glial cells in vivo after engraftment to the adult injured spinal cord. Further in vivo studies are necessary to find out the extent of the neuronal and glial subpopulations, the possibility to regulate the differentiation into certain phenotypes, as well as investigate whether the human neuronal cells are establishing functional connections with the host.
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Acknowledgments We thank Prof. S. Pfeiffer, University of Connecticut, CT, U.S.A. for his kind supply of O4 antibodies. This study was generously supported by Craig Hospital, Denver, CO, Hjärnfonden (Swedish Brain Foundation), The Research Funds of Karolinska Institutet, The Swedish Research Council (14X-06555) and The Stockholms Sjukhem Foundation. References [1] Zhang S-C, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–33. [2] Carpenter MK, Cui X, Hu Z-Y, Jackson J, Sherman S, Seiger Å, et al. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 1999;158:265–78. [3] Svendsen CN, ter Borg MG, Armstrong RJE, Rosser AE, Chandran S, Ostenfeld T, et al. A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods 1998;85:141–52. [4] Kukekow VG, Laywell ED, Suslov O, Davies K, Scheffler B, Thomas LB, et al. Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol 1999;156: 333–44. [5] Bixby S, Kruger GM, Mosher JT, Joseph NM, Morrison SJ. Cellintrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 2002;35:643–56. [6] Enomoto M, Shinomiya K, Okabe S. Migration and differentiation of neural progenitor cells from two different regions of embryonic central nervous system after transplantation into the intact spinal cord. Eur J Neurosci 2003;17:1223–32. [7] Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature 2002;417:39–44. [8] Cao Q-L, Zhang YP, Howard RM, Walters WM, Tsoulfas P, Whittemore SR. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to glial lineage. Exp Neurol 2001;167:48–58. [9] Cao Q-L, Howard RM, Dennison JB, Whittemore SR. Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord. Exp Neurol 2002;177:349–59. [10] Quinn SM, Walters WM, Vescovi AL, Whittemore SR. Lineage restriction of neuroepithelial precursor cells from fetal human spinal cord. J Neurosci Res 1999;57:590–602.
[11] Walder S, Ferreti P. Distinct neural precursors in the developing human spinal cord. Int J Dev Biol 2004;48:671–4. [12] England MA. A colour atlas of life before birth. Aylesbury, Bucks, England: Hazell books; 1990. [13] Gabay L, Lowell S, Rubin LL, Anderson DJ. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 2003;40:485–99. [14] Odeberg J, Piao J-H, Samuelsson E-B, Falci S, Åkesson E. Low immunogenicity of in vitro expanded human neural cells despite high MHC expression. J Neuroimmunol 2005;161:1–11. [15] Johansson CB, Momma S, Clarke DL, Riesling M, Lendahl U, Frisén J. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 1999;96:25–34. [16] Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, et al. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 1996;16:7599–609. [17] Nunes MC, Roy NS, Keyoung HM, Goodman RR, McKhann II G, Jiang L, et al. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med 2003;9:439–47. [18] Westerlund U, Moe MC, Varghese M, Berg-Johnsen J, Ohlsson M, Langmoen IA, et al. Stem cells from the adult human brain develop into functional neurons in culture. Exp Cell Res 2003;289:378–83. [19] Svendsen CN, Skepper J, Rosser AE, ter Borg MG, Tyres P, Ryken T. Restricted growth potential of rat neural precursors as compared to mouse. Dev Brain Res 1997;99:253–8. [20] Ostenfeld T, Joly E, Tai Y-T, Peters A, Caldwell M, Jauniaux E, et al. Regional specification of rodent and human neurospheres. Dev Brain Res 2002;134:43–55. [21] Hitoshi S, Tropepe V, Ekker M, van der Kooy D. Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain. Development 2002;129:233–44. [22] Chow SY, Moul J, Tobias CA, Himes BT, Liu Y, Obrocka M, et al. Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord. Brain Res 2000;874: 87–106. [23] Shihabuddin LS, Horner PJ, Ray J, Gage FH. Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci 2000;20:8727–35. [24] Wu S, Suzuki Y, Kitada M, Kitaura M, Kataoka K, Takahashi J, et al. Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord. Neurosci Lett 2001;312:173–6. [25] Svendsen CN, Caldwell MA, Ostenfeld T. Human neural stem cells: isolation, expansion and transplantation. Brain Pathol 1999;9:499–513.
Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres Elisabet Åkesson a,d,⁎, Jing-Hua Piao a,d , Eva-Britt Samuelsson a , Lena Holmberg a , Anders Kjældgaard b , Scott Falci c , Erik Sundström a,d , Åke Seiger a,d a
Karolinska Institutet, Division of Neurodegeneration and Neuroinflammation, Department of Neurobiology, Care Sciences and Society, Novum, S-141 86 Stockholm, Sweden b Karolinska Institutet, Division of Obstetrics/Gynecology, Department of Clinical Sciences, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden c Craig Hospital, 3425 S. Clarkson St., Englewood, CO 80110, USA d Stockholms Sjukhem Foundation, Mariebergsgatan 22, Stockholm, Sweden
Abstract There is heterogeneity in neural stem and progenitor cell characteristics depending on their species and regional origin. In search for potent in vitro-expanded human neural precursor cells and cell therapy methods to repair the injured human spinal cord, the possible influence exerted by intrinsic cellular heterogeneity has to be considered. Data available on in vitro-expanded human spinal cord-derived cells are sparse and it has previously been difficult to establish long-term neurosphere cultures showing multipotentiality. In the present paper, human spinal cord-derived neurospheres were cultured in the presence of EGF, bFGF and CNTF for up to 25 passages (N350 days) in vitro. In contrast to the human first trimester subcortical forebrain, spinal cord tissue N 9.5 weeks of gestation could not serve as a source for long-term neurosphere cultures under the present conditions. After withdrawal of mitogens, cultured neurospheres (at 18 passages) gave rise to cells with neuronal, astrocytic and oligodendrocytic phenotypes in vitro. After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immunodeficient adult rats, large numbers of cells survived at least up to 6 weeks, expressing neuronal and astrocytic phenotypes. These results demonstrate that it is possible to expand and maintain multipotent human spinal cord-derived neurospheres in vitro for extended time-periods and that they have promising in vivo potential after engraftment to the injured spinal cord. © 2007 Elsevier Inc. All rights reserved. Keywords: Human; Embryonic; Spinal cord; Forebrain; Neurospheres; Culture; PCNA; Transplantation; Spinal cord injury
1. Introduction The possibility to expand human neural stem cells in vitro has increased the availability of potential donor cells for transplantation intervention after nervous system injury and disease. Multipotent human neural stem cells may be generated from embryonic stem (ES) cells [1] embryonic and fetal [2,3] as well as adult nervous system tissue [4]. However, multiple donor and host factors such as developmental stage, central nervous system (CNS) region and species-specific characteristics may in⁎ Corresponding author. Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Novum 5th floor, S-141 86 Stockholm, Sweden. Tel.: +46 8 58583907; fax: +46 8 58583880. E-mail address: [email protected] (E. Åkesson). 0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.05.056
fluence the in vitro and in vivo potential of neural stem cells [2,5–7]. This underlines the importance of using experimental models that mimic the clinical conditions for evaluation of the human donor cell and host properties as well as the transplantation procedure used. To repair the injured spinal cord it is of importance to evaluate the spinal cord-specific properties of the potential host and donor cells. With regard to the host, the adult spinal cord has been reported as non-neurogenic, not supporting neurogenesis of neural precursor cell transplants in allograft rodent models [8–10]. In vitro studies by Song and collaborators showed that astrocytes from adult rodent hippocampus promote neurogenesis while astrocytes from adult rodent spinal cord do not [7]. Furthermore, the conditions in the injured adult spinal cord were shown to be even less permissive than those of the normal spinal
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cord [9]. With regard to the donor cells, little is known about spinal cord-derived stem/progenitor cells. Few reports have focused on in vitro expansion of human spinal cord-derived neural precursor cells [10,11] and only one has previously shown stable long-term in vitro expansion of human spinal cord-derived neurospheres [10]. These neurospheres were shown to include only glial lineage-restricted precursor cells after differentiation. The aim of the present study was to expand multipotent human spinal cord-derived neurospheres for intraspinal transplantation. We here report successful long-term in vitro expansion of multipotent human spinal cord-derived neurospheres in the presence of ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). In addition, we found that human spinal cord-derived neural stem and progenitor cells survive and present both neuronal and glial phenotypes in vivo after engraftment to adult injured rodent spinal cord. 2. Methods and procedures 2.1. Human first trimester spinal cord and forebrain tissue Human first trimester subcortical forebrain (in the following text also referred to as forebrain) and spinal cord tissues (4.5– 12 weeks of gestation) were collected after elective routine abortions using gentle vacuum aspiration technique. Informed consent was obtained from the pregnant women. The collection was approved by the Human Ethics Committee of Huddinge University Hospital. The gestational age of the tissue was determined by examination of anatomical landmarks and the size of the abortion material according to the atlas of England [12]. The subcortical forebrain and spinal cord were dissected and freed of meninges with sterile technique in DMEM: F12 medium (Life Technologies) and divided into two pieces. One part was fixed, cryo-sectioned and prepared for immunohistochemical analyses (see below) to compare the presence of proliferating neural cells in the in situ first trimester subcortical forebrain and spinal cord tissues. The remaining part of the tissue was processed for neurosphere culture. In previous reports human fetal forebrain more frequently than spinal cord tissue has served as source for neurosphere culture. Therefore we compared the outcome of cultures from the two regions. 2.2. Human spinal cord- and forebrain-derived neurosphere cultures The tissue samples were mechanically dissociated into single cell suspensions by the use of a glass-Teflon homogenizer. The method we used for establishing and propagating neurospheres is a slight modification of the protocol developed by Carpenter and co-workers [2]. Cells were cultured in 75 ml non-coated flasks (Nunc) with 20 ml of cell suspension at a density of 100,000–150,000 cells/ml medium (DMEM: F12 at 1:1, Life Technologies) supplemented with 0.6% glucose, 5 mM Hepes, Heparin (2 μg/ml, Sigma), 1% N2-supplement (v/v, Life Technologies), EGF (20 ng/ml, R&D Systems), bFGF (20 ng/ml, R&D systems) and CNTF (10 ng/ml, R&D Systems) to form
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free-floating neurospheres. The cells were maintained at 37 °C in 5% CO2. Fresh medium was added twice a week. The neurospheres were passaged every 7–20 days (depending on the growth of the individual culture) by mechanical dissociation and then re-cultured in fresh medium as described above. All the neurosphere cultures were performed without antibiotics. For in vitro differentiation, the human neurospheres were mechanically dissociated by trituration and seeded on poly-Dlysine (Sigma, 0.1 mg/ml) and fibronectin-coated (0.1 mg/ml, Sigma) circular glass cover slips (13 mm in diameter) in 24-well plates (Nunc). The cells were seeded at a density of 26,000 cells/ ml DMEM: F12 supplemented with 0.6% glucose, 5 mM Hepes, 1% N2-supplement and 10% fetal calf serum, and cultured for 7 days prior to fixation and immunocytochemistry. To in vitro differentiate oligodendrocytes from human neurospheres a modified protocol developed by Zhang and co-workers (Zhang et al., 2000) was used, with mechanical dissociation and seeding in medium as above but in 0.5% fetal calf serum instead of 10% and the addition of 1 ng/ml recombinant human PDGF-AA (PeproTech Inc.). The medium was changed every 3 days during the 4 weeks of culture prior to fixation and immunocytochemistry. 2.3. Intraspinal transplantation of human spinal cord-derived neurospheres A spinal cord compression lesion was performed in 12 adult athymic female rats (HsdHan: RNU-rnu, Harlan, UK) weighing 170–200 g. The rats received intraperitoneal (i.p.) injections of atropine preoperatively (Atropine, 0.05 mg/kg, NM Pharma AB, Sweden) followed by i.p. injections of phenyl citrate, 0.22 mg/kg and fluanisone, 6.75 mg/kg (Hypnorm, Janssen Pharmaceutical) combined with midazolam (3.4 mg/kg, Dormicum, Roche). A partial laminectomy was made in the lower half of vertebrae L1 and a sagittal cut was made in the dura mater. Two drops of lidocain (20 mg/ml Xylocain, Astra Zeneca) was added to the exposed spinal cord just prior to the compression. The compression lesion was made by the use of a bulldog clamp with a closing pressure of 35 g (De Bakey, 58 mm in total length) applied vertically over the spinal cord for 30 s. The lesioned spinal cord was covered with one layer of meningeal substitute (Lyoplant, B/Braun Aesculap) prior to suture of the wound. Per-operative body temperature was controlled by the use of heating pads with a rectal thermometer (CMA/150, Carnegie Medicine). For post-surgical analgesia the animals were given buphrenorphine (Temgesic, 7 μg/kg, Reckitt & Colman) intramuscularly twice a day for 4 days. The urinary bladders were emptied manually twice a day. To prevent urinary infections the rats were given antibiotics (Borgal vet., 15 mg/kg, Hoechst) subcutaneously 4 days per week. Two weeks after the spinal cord compression lesions, the animals were re-anaesthetized and the lesioned area carefully exposed. Human neurospheres at passage four (P4), deriving from a 6.5–7 gestational week old spinal cord, 10–12 in number with a diameter ranging from 0.1 to 0.25 mm were injected through a glass-capillary into the spinal cord parenchyma close to the lesion area in 8 of the 12 lesioned cases. The remaining four animals underwent the same surgical procedure but with
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intraspinal injections of medium lacking neurospheres, serving as controls. After a post-surgical/transplantation period of 6 weeks, the rats were re-anesthetized once more and perfused through the ascending aorta with 100 ml calcium-free Tyrode's solution followed by 400 ml of 4% para-formaldehyde in phosphate buffer, pH 7.4. The dissected spinal cords were postfixed for another 90 min, rinsed and kept in 30% sucrose until they were cryostat-sectioned at 10 μm. The experimental procedure was approved by “The Experimental Animal Ethics Committee” of Southern Stockholm.
EDTA, 0.1% sodium dodecyl sulphate, 1% Triton X-100, Complete proteinase inhibitor (Roche) and 50 mM Tris–HCl (pH 7.6). Proteins were separated by electrophoresis on a 10% Tris– glycine sodium dodecyl sulphate-polyacrylamide gel, transferred to a nitrocellulose filter and incubated with primary antibodies towards PCNA (1:500, Santa Cruz Biotechnology). After detection of primary antisera with anti-rabbit immunoglobulin-horseradish peroxidase, the immunoblots were developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
2.4. Immunohistochemical analyses
3. Results
Formaldehyde-fixed human first trimester forebrain and spinal cord tissue was cryo-sectioned and treated with 5% acetic acid/95% ethanol (previously cooled to −20 °C) for 10 min. The sections were incubated with 1.5% normal goat serum for 30 min prior to incubation over-night with primary antiserum to proliferating cell nuclear antigen (PCNA, 1:10, Santa Cruz Biotechnology). The in vitro-differentiated human neurosphere cells were fixed with buffered 4% para-formaldehyde pH 7.4 for 10 min and rinsed in phosphate buffered saline (PBS). To reduce background the cells were incubated in 10% normal goat serum in PBS for 30 min prior to addition of the primary antisera to βtubulin III (1:800, Sigma), glial fibrillary acidic protein (GFAP, 1:500 DAKO), galactocerebroside C (GalC, 1:50, Chemicon) or O4 (1:50, gift from Dr. Pfeiffer, Univ. of Connecticut, CT). Spinal cord sections with or without transplants were incubated over-night with monoclonal antibodies to human nuclear protein (HNP, 1:250, Chemicon) and glial fibrillary acidic protein (GFAP, 1:20,000, Sternberger Monoclonals Inc.) as well as the following polyclonal antibodies raised against β-tubulin III (1:1,200, Berkeley Antibody Company), and Gal C (1:50, Chemicon) to analyze the cellular phenotypes of grafted human spinal cord-derived neurospheres. All primary antisera were diluted in 0.1 M PBS with 0.3% Triton X-100. After incubation with primary antisera over-night at + 4 °C, the sections/cells were rinsed in PBS for 3 × 10 min. Detection of primary antibodies was performed with secondary antibodies conjugated to Alexa Fluor® 488 (1:400, Molecular Probe) or Cy3 (1:800, Jackson Immunoresearch Laboratories, Inc.) added and incubated for 60 min followed by a final rinse in PBS for 3 × 10 min. Gal C immuno-labeling was visualized using a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (EnVision+™, Peroxidase, DAKO). Negative control slides were processed as above, but the primary antisera were omitted. Furthermore, extra slides with tissue sections including human neurons, astrocytes and oligodendrocytes were processed in parallel as positive controls. The images were viewed with a fluorescence microscope (Zeiss, Axiophot) and acquired using a CCD camera (Hamamatsu, ORCA-ER, C4742-95) and the Openlab software for Macintosh (Improvision).
3.1. Long-term culture of human subcortical forebrain- and spinal cord-derived neurospheres
2.5. Immunoblot Human first trimester spinal cord tissue, 5, 7, 9.5 or 10.5 weeks of gestation, were homogenized in lysis buffer comprising 2 mM
Cells isolated from human forebrain 4.5–12 weeks of gestation grew as free-floating neurospheres in the presence of EGF, bFGF and CNTF. The success rate of human neurosphere cultures, at P2, P5 and P10, with different gestational ages is summarized in Table 1. Forebrain-derived cultures could be established and sustained even from late first trimester stages. A majority of the cultures showed at least a five-fold increase in cell number at P10 compared to that at P0. The culture that expanded the most showed a 267-fold increase in cell number up to P10. A neurosphere culture deriving from forebrain tissue at 12 weeks of gestation was sustained up to at least P18. The forebrain neurospheres that were kept in culture the longest, up to P25, originated from a 5 week old embryo. Cells from human first trimester spinal cord formed neurospheres and increased in number, as long as the gestational age of the original tissue was ≤9.5 weeks of gestation. Neurospheres derived from 4.5–9.5 gestational week spinal cord could be expanded long-term, while donor stages beyond 9.5 weeks of gestation could not be propagated for more than four passages (Table 1). All the spinal cord-derived cultures, sustained up to P10, showed an increase in cell number compared to that at P0. Most of these expanded more than five-fold. The culture that expanded the most showed a 200-fold increase in cell number up to P10. The spinal cord-derived neurospheres Table 1 Human spinal cord- and forebrain-derived neurosphere cultures Gestational age
Passage (P)
Spinal cord n /n
%
ns/nt
%
≤8 weeks
2 5 10 2 5 10 2 5 10
42/45 27/31 11/15 6/8 3/6 2/5 2/6 0/5 0/5
93 87 73 75 50 40 33 0 0
42/44 18/29 12/24 5/6 4/6 4/6 8/12 4/11 3/10
95 62 50 83 75 75 67 36 30
8.5–9.5 weeks
N9.5 weeks
s
t
Forebrain
Summary over human spinal cord- and forebrain-derived cultures that resulted in sustained neurosphere formation at different donor stages and passage number (P). ns, number of sustained neurosphere cultures; nt, total number of neurosphere cultures.
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that were kept in culture the longest, up to P25 (N 350 days in culture), originated from a 7 week old embryo. 3.2. Cellular phenotypes in vitro and following intraspinal transplantation To study the cellular phenotypes of long-term cultured human neurospheres, dissociated cells were plated on cover slips and cultured without CNTF, EGF and bFGF. Both longterm cultured human forebrain- and spinal cord-derived neuro-
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spheres showed numerous β-tubulin III- and GFAP-immunoreactive (IR) cells. Gal C- and O4-IR cells were also observed but were sparse in number (Fig. 1A and B). After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immuno-deficient adult rat, numerous surviving HNP-IR cells were observed in five of seven grafted cases at 6 weeks after the graft injection, indicating human cell survival. One of the transplanted immunedeficient rodents was sacrificed prior to the endpoint due to infection and was not included in the study. No HNP-IR cells
Fig. 1. In vitro and in vivo cellular phenotypes of human spinal cord-derived neurosphere cells. A) After 18 passages in vitro, human neurospheres deriving from human spinal cord tissue, 7 weeks of gestation, were differentiated without mitogens but with fetal calf serum present in the culturing medium. GFAP- (red) and β-tubulin-IR cells (green) were numerous while B) Gal C- and O4-(see inset) IR cells were sparse in number. Scale bar = 20 μm. C) After intraspinal transplantation of human spinal cord-derived neurospheres numerous human cells including both neuronal and glial phenotypes survived in the compressionlesioned adult rat spinal cord. HNP-IR nuclei (green) with varying size and shape surrounded by nestin-IR (red) in the epicenter of transplant. D) Cells that had migrated approximately 400 μm from the transplantation site into host spinal cord showed HNP-IR nuclei (green) surrounded by nestin-IR cytoplasm and extensions (red). E) HNP- (green) and β-tubulin-IR cells (red) suggesting that the cell has neuronal phenotype. F) HNP- (green) and GFAP-IR cells (red) suggesting that these cells have astrocytic phenotypes.
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Fig. 2. Proliferating cells in in situ human forebrain and spinal cord at 7 and 10.5–11 weeks of gestation. PCNA-IR cells in the ventricular zone of 7 (A) and 10.5–11 gestational weeks old forebrain (B) and surrounding the immature central canal in the spinal cord at 7 weeks of gestation (C). The number of PCNA-IR cells was much lower in the spinal cord at 10.5–11 weeks of gestation (D) compared to that at 7 weeks of gestation. Bars = 100 μm. PCNA immunoblot of human spinal cord tissue 5– 10.5 weeks of gestation (E). The PCNA-IR bands had a molecular weight of approximately 36 kDa.
were observed in non-transplanted spinal cords. In grafted cases the vast majority of the observed HNP-IR cells also had a nestin-IR cytoplasm, suggesting that most of the grafted human cells were still neural precursors (Fig. 1C and D). However, in adjacent sections multiple cells were double-labeled with βtubulin III and HNP (Fig. 1E) or GFAP and HNP (Fig. 1F) suggesting that a subpopulation of the human-derived cells had differentiated into neuronal and glial phenotypes. The β-tubulin III-IR cells were frequently localized in groups closer to the implantation site, while the GFAP-IR cells were more scattered and seemed to have migrated further at this point in time. In contrast, no Gal C-IR was observed to be co-localized with any HNP-IR nuclei, suggesting that no grafted human neural precursor cells had differentiated into oligodendrocytes. 3.3. Proliferating cells in first trimester human CNS tissue In view of the decline in neurosphere formation and survival from the spinal cord with increasing gestational age, possible changes in the proliferating cell population of the source tissue were investigated. The presence of proliferating cells in situ in the human spinal cord and subcortical forebrain (5 to 11 weeks of gestation) was investigated by the use of an immunohistochemical marker for PCNA. We hypothesized that the decline in neurosphere formation coincides with the decrease of proliferative cells in the ventricular zone. In the subcortical forebrain
from which we readily could establish neurospheres at least as late as 12 weeks of gestation, a large number of PCNA-IR cells were observed at 7 and 10.5–11 weeks of gestation (Fig. 2A and B). On the other hand, in the in situ human spinal cord PCNA-IR cells which were abundant in the neuroepithelial cell layer at 7 weeks of gestation (Fig. 2C), were lacking in six and clearly reduced in two of the investigated eight cases at 10– 11 weeks of gestation (Fig. 2D), a fetal stage when neurospheres no longer could be established using the present protocol. Immunoblot confirmed the decline in PCNA-IR in the human spinal cord tissue from 5 to 10.5 weeks of gestation (Fig. 2E). 4. Discussion In the present study we demonstrate long-term in vitro cultures of human spinal cord-derived neurospheres with the potential to differentiate into neuronal, astrocytic and oligodendrocytic phenotypes. The long-term expansion of multipotent human spinal cord neural cells will allow us to compare their in vitro and in vivo potential with that of neural cells from other regions and other cellular sources in spinal cord repair models. Long-term maintenance of human spinal cord-derived multipotent neurospheres has previously not been described in detail according to our knowledge. Quinn et al. [10] succeeded with two cultures in the presence of EGF and bFGF up to at least 25 passages. However, in contrast to our results these cultures only
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gave rise to astrocytes suggesting that the culture conditions used selected for glial precursors. The question whether tripotent neural stem cells exist in vivo has been raised and Gabay and collaborators showed that clonogenic competence to generate neurons, astrocytes and oligodendrocytes was induced by FGF-2 deregulation of dorso-ventral patterning during expansion in vitro [13]. This induction of tripotency may occur as a result of the bFGF, EGF and CNTF presence, also in our human neurosphere cultures. The aim of cellular transplantation to a spinal cord lesion is to substitute for the extensive cell loss, be neuroprotection and support of remyelination and neuronal regeneration, in order to reach physiological restitution. Traumatic spinal cord injury (SCI) results in loss of multiple cell types. Cell therapy would preferentially include a cell population with both neuronal and glial potential. Whether a homogenous population of multipotent stem cells, a heterogeneous population of more restricted precursor cells, or a mixture of those cell types would be optimal for SCI repair is still under investigation. However, for clinical treatment it is necessary that these cells can be expanded in large numbers, maintained in vitro for months and that they remain stable with regard to differentiation potential. We suggest that human embryonic spinal cord neurosphere cultures may have this potential although further characterization of the cells is crucial to ensure stability. We have previously characterized human spinal cord-derived neurospheres concerning their immune-competence, of relevance for cell therapy application. The in vitro-expanded human neural precursor cells expressed high levels of MHC proteins on their cell surface but still did not elicit an immune response in co-cultures with peripheral human lymphocytes [14]. Using the present culturing method we succeeded in establishing and maintaining multipotent human spinal cord neurospheres long-term from donors ≤ 9.5 weeks of gestation. Beyond that developmental stage no spinal cord-derived neurospheres could be maintained for more than four passages. In the human forebrain cultures, on the other hand, we could expand neurospheres even from a case at 12 weeks of gestation. It is known, that neural stem cells remain along the entire neuraxis including the adult mammalian spinal cord [15,16]. However, despite that neurosphere formation has been reported from adult human brain cells [4,17,18] there is no report showing in vitro neurosphere formation from the adult human spinal cord. The lack of long-term neurosphere cultures from later first trimester spinal cord tissue in our hands is in concordance with the lack of initial neurosphere formation in second trimester human spinal cord specimens reported by Quinn et al. [10]. We hypothesized that human neurospheres are derived from proliferating neural precursor cells in the immature central nervous tissue. PCNA immunocytochemistry indicated that the number of dividing cells in the human spinal cord was lower at 10–11 weeks compared to that at 7 weeks of gestation, while still abundant in the subcortical forebrain at 10.5 weeks of gestation. This is in concordance with the observation of no long-term spinal cord-derived cultures beyond 9.5 weeks of gestation, while successful long-term cultures from late first
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trimester forebrain could be obtained. However, the fact that the number of PCNA-IR cells is approximately 1000 times larger than the number of forming neurospheres in vitro at the same embryonic stages (data not shown) suggests that it may be a subpopulation of the PCNA-IR cells that form the neurospheres. To propagate the human neurospheres we used the mitogens EGF and bFGF but also added the interleukin-6 family member CNTF in our cultures from initial seeding. Human forebrainderived neurosphere cultures were previously expanded in the presence of EGF, bFGF and human leukemia inhibitory factor (hLIF). Cultures grown with addition of hLIF (10 ng/ml) consistently showed an enhanced proliferation after 50–60 days in vitro, as well as an increased neuronal population after differentiation compared to equivalent cultures grown with EGF and bFGF alone [2]. For human forebrain-derived neurospheres, hLIF could be substituted with CNTF [2]. There are both interspecies and regional differences in the way neural precursor cells are regulated [5,19–21]. These factors need to be taken into account when attempting to standardize in vitro neural cell expansion conditions. With the aim to repair the injured spinal cord in man it is important to study conditions specifically necessary for expansion of human neural cells, and how they interact with the injured adult spinal cord after transplantation. Differentiation of rodent neural stem/ progenitor cells transplanted into the adult spinal cord was previously described as mainly restricted to the glial lineage [6,8,22–24]. Even neuronally restricted rodent precursor cells were inhibited to differentiate into neuronal phenotype when grafted into the contused adult rat spinal cord [9]. In the present study numerous human spinal cord-derived cells survived the transplantation to the lesioned immuno-deficient adult rat spinal cord with a large population being nestin-IR and subpopulations showing GFAP- and β-tubulin III-IR. Our human neural precursor cells showing tripotency in vitro were here shown to have the ability to differentiate into neurons and astrocytes but not oligodendrocytes (with Gal C as the immunohistochemical marker) after transplantation to the injured spinal cord. The differentiation of oligodendroglia from human fetal brainderived neurospheres, reported by others, has been limited and most oligodendroglia remain at the relatively immature O4+-stage, preceding the Gal C+-stage [1,25] that may explain why we did not observe any Gal C-IR human cells in vivo after transplantation. 5. Concluding remarks The present report demonstrates long-term in vitro expansion of human spinal cord-derived neurospheres including precursor cells with the potential to develop into astrocytes, neurons and oligodendrocytes. These neurosphere-derived spinal cord cells may in turn survive, migrate and express phenotypic proteins for neurons and glial cells in vivo after engraftment to the adult injured spinal cord. Further in vivo studies are necessary to find out the extent of the neuronal and glial subpopulations, the possibility to regulate the differentiation into certain phenotypes, as well as investigate whether the human neuronal cells are establishing functional connections with the host.
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Acknowledgments We thank Prof. S. Pfeiffer, University of Connecticut, CT, U.S.A. for his kind supply of O4 antibodies. This study was generously supported by Craig Hospital, Denver, CO, Hjärnfonden (Swedish Brain Foundation), The Research Funds of Karolinska Institutet, The Swedish Research Council (14X-06555) and The Stockholms Sjukhem Foundation. References [1] Zhang S-C, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–33. [2] Carpenter MK, Cui X, Hu Z-Y, Jackson J, Sherman S, Seiger Å, et al. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 1999;158:265–78. [3] Svendsen CN, ter Borg MG, Armstrong RJE, Rosser AE, Chandran S, Ostenfeld T, et al. A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods 1998;85:141–52. [4] Kukekow VG, Laywell ED, Suslov O, Davies K, Scheffler B, Thomas LB, et al. Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol 1999;156: 333–44. [5] Bixby S, Kruger GM, Mosher JT, Joseph NM, Morrison SJ. Cellintrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 2002;35:643–56. [6] Enomoto M, Shinomiya K, Okabe S. Migration and differentiation of neural progenitor cells from two different regions of embryonic central nervous system after transplantation into the intact spinal cord. Eur J Neurosci 2003;17:1223–32. [7] Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature 2002;417:39–44. [8] Cao Q-L, Zhang YP, Howard RM, Walters WM, Tsoulfas P, Whittemore SR. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to glial lineage. Exp Neurol 2001;167:48–58. [9] Cao Q-L, Howard RM, Dennison JB, Whittemore SR. Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord. Exp Neurol 2002;177:349–59. [10] Quinn SM, Walters WM, Vescovi AL, Whittemore SR. Lineage restriction of neuroepithelial precursor cells from fetal human spinal cord. J Neurosci Res 1999;57:590–602.
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