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First published online July 28, 2005
Stem Cells Vol. 23 No. 9 October 2005, pp. 1295 -1303
doi:10.1634/stemcells.2005-0026; www.StemCells.com
© 2005 AlphaMed Press

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Transplantable Neural Progenitor Populations Derived from Rhesus Monkey Embryonic Stem Cells

Tianqing Lia,b,c, Jiawei Zhengc,d, Yunhua Xiea,b, Shufen Wanga,b,c, Xiuzhen Zhanga,b,c, Jian Lia, Lifang Jina,b,c, Yuanye Mab,d, Don P. Wolfe, Qi Zhouf, Weizhi Jiaa,b

a Department of Reproduction and Development, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China;
b Kunming Primate Research Center, The Chinese Academy of Sciences, Kunming, Yunnan, China;
c Graduate School, The Chinese Academy of Sciences, Beijing, China;
d Section of Cognitive Brain Research, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China;
e Oregon National Primate Research Center, Portland, Oregon, USA;
f Institute of Zoology, The Chinese Academy of Sciences, Beijing, China

Key Words. Rhesus monkey embryonic stem cells • Neural progenitors • Hepatocyte growth factor • G5 supplement • Differentiation

Correspondence: Weizhi Ji, Ph.D., Kunming Primate Research Center and Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan, 650223, China. Telephone: 86-871-5139413; Fax: 86-871-5139413; e-mail: wji{at}mail.kiz.ac.cn; and Qi Zhou, Institute of Zoology, The Chinese Academy of Sciences, Beijing 100086, China. Telephone: 86-10-62650042; e-mail: qzhou{at}ioz.ac.cn


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3% ± 8.1%) populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65%–80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The isolation of embryonic stem cells (ESCs) has generated interest in their potential use in cell replacement therapies for degenerative diseases and as models of cell development and differentiation. Currently, a major effort is focused on defining in vitro conditions for inducing ESC differentiation into specific cell types required for clinical therapies.

Previous studies have shown that ESCs can be induced to generate primitive neural stem cells (NSCs) in chemically defined low-density culture conditions [1] or in the presence of stromal cell–derived inducing activity (SDIA) [2, 3]. However, only 0.1%–0.2% of ESCs differentiate into neural progenitors (NPs) [1], and the biological significance of SDIA for neurogenesis is unclear [2–4]. Additionally, Lee and colleagues efficiently obtained NPs by culture in a selective, serum-free medium containing basic fibroblast growth factor (bFGF) to eliminate non-neural cells [5]. In humans and monkeys, neural precursors have been obtained from ESCs [6, 7]. However, a dramatic decrease in cell number occurs, as the majority of the cells do not survive the culture conditions [8], albeit in low yields; for example, the percentage of neural precursors detectable in differentiated embryoid bodies (EBs) derived from ESCs was less than 80% [6, 7]. Therefore, defining culture conditions that more efficiently induce ESC differentiation into neural precursors is a precondition for further progress in this field.

Hepatocyte growth factor (HGF) is a pleiotrophic cytokine that can trigger the proliferation, migration, and differentiation of various cell types [9, 10]. Increasing evidence suggests that HGF and its receptor, c-Met, are expressed in the adult and developing nervous system [11, 12] and that HGF plays an important role in the nervous system [12, 13]. These observations suggest that HGF may induce a functional response in neural populations in the developing or adult central nervous system. We also developed an interest in G5 supplement (Gibco, Grand Island, NY, http://www.invitrogen.com) based on the manufacturer’s suggestion that this supplement was appropriate for the growth and expression of glial or astrocytic phenotypes [14].

Here, we used defined medium containing HGF and G5 supplement to induce rhesus monkey ESC (rESC) differentiation into transplantable populations of NPs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of ESCs
R366.4 rESCs (a gift from Dr. James Thomson) were cultured on a feeder layer of irradiated MESFs (ear skin fibroblasts from a 1-week-old rhesus monkey) [15] in ESC culture medium [16].

Production and Culture of NPs from rESCs
Prior to differentiation, rESC colonies were digested with 1 mg/ml dispase, washed to remove dispase, suspended in the ESC culture medium, and plated in six-well plates containing feeder cells that had been allowed to attach for 6 hours. After this incubation, rESCs remaining in the medium were collected by centrifugation, resuspended in differentiation medium (Dulbecco’s modified Eagle’s medium [DMEM]/F12, 15% fetal bovine serum [FBS] [HyClone, Logan, UT, http://www.hyclone.com], 2 mM glutamine, 0.1 mM ß-mercaptoethanol, and x1 nonessential amino acids), and cultured in hanging drops for EB production (30 µl/drop, 60 cells/µl). Nine-day-old EBs (3 days in hanging drops and 6 days in suspension) were then plated onto 0.5% gelatin-coated culture plates in neural differentiation culture media (NDCM) (DMEM/F12 supplemented with 1xITS [Gibco], 1.0 g/L bovine serum albumin, 1.0 g/L glucose, 1.0 g/L lactose, 0.03 g/L proline, 11 µg/L linoleic acid, 5 mM glutamine, and 2 mM nicotinamide) in the presence of G5 supplement (x1; Gibco) (composition: insulin 500 µg/ml, human transferrin 5 mg/ml, selenite 0.52 µg/ml, biotin 1 µg/ml, hydrocortisone 0.36 µg/ml, FGF2 0.52 µg/ml, and epidermal growth factor [EGF] 1 µg/ml) and 10 ng/ml HGF (Chemicon International, Temecula, CA, http://www.chemicon.com). Six experimental groups were evaluated: (a) basic medium, NDCM; (b) bFGF (10ng/ml) + NDCM, (bFGF control) [5–7]; (c) HGF + NDCM, (HGF); (d) G5 supplement + NDCM, (G5); (e) HGF + G5 supplement + NDCM, (HGF+G5); and (f) serum (10% FBS) + HGF+G5 + NDCM, (S+HGF+G5). After 10 days of culture, Hoechst 33342 and nestin positive differentiated cells were counted.

For the isolation and purification of NPs, differentiated cells in EBs were digested with trypsin (0.125% in 0.04% EDTA) until small-elongated cells in the dispersed population rounded up. These cells were manually agitated to complete the dissociation process and replated on new gelatin-coated plates. Cell aliquots were stained with Hoechst 33342 and nestin to quantify the efficiency of the NPs’ purification.

Expansion and Differentiation of NPs into Neural Lineages
Dispersed NPs were expanded in monolayer cultures or as neurospheres in NDCM plus G5 supplement, or HGF+G5 supplement. Monolayer cells or neurospheres were digested with trypsin (0.125% in 0.04% EDTA) in preparation for passaging. To analyze the differentiation potential of ESC-derived NPs, cells were cultured on 0.5% gelatin or 20 µg/ml laminin (Chemicon International)–coated plates in NDCM in the absence of HGF and G5 supplement.

NP Subcloning
NPs from the HGF+G5 group cultured for 6 weeks were dispersed into single cells with 0.125% trypsin, selected manually with the micromanipulator under phase-contrast microscopy, transferred into microdrops on gelatin-coated plastic (100 µl), and cultured in NDCM with 10 ng/ml HGF and G5 supplement covered with mineral oil. Confluent cells (after 7–14 days) were digested with trypsin and passaged in NDCM containing HGF and G5 supplement. Clonally derived NP lines were induced to differentiate by removal of HGF and G5 supplement. After 3 weeks, differentiated cells were subjected to immunocytochemical (ICC) staining.

ICC Staining
Standard protocols were used for ICC staining of neural spheres, disaggregated progenitor cells, and differentiated cells. In general, cells were fixed in situ with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at room temperature. Incubation for 10 minutes with 0.2% Triton X-100 was preceded by two washes with PBS. After blocking with 10% goat serum, the cells were stained with one of the following primary antibodies: nestin monoclonal antibody, O4 monoclonal antibody, glial fibrillary acidic protein (GFAP) polyclonal antibody, microtubule-associated protein 2 (MAP2) polyclonal antibody, serotonin monoclonal antibody, or neuronal nuclear antigen (NeuN) monoclonal antibody (all from Chemicon International), myelin basic protein (MBP) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), or synaptophysin polyclonal antibody (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us). The cells were then rinsed three times with PBS and incubated for 30 minutes with the corresponding goat anti-fluorescein isothiocyanate– or phycoerythrin-conjugated second antibody (Santa Cruz Biotechnology). Negative controls for each fluorophore-conjugated secondary antibody, carried out without the addition of the primary antibody, were included to evaluate nonspecific binding of secondary antibodies. Approximately 900 immunolabeled cells were examined in each experiment, using a confocal laser scanning system (LSM 510 META; Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Intracerebroventricular Transplantation and In Vivo Analysis
To evaluate the behavior of NP populations derived from rESCs in vivo, adult Sprague-Dawley rats (190–210 g) were used. Experimental protocols met the guidelines of the Institute’s Internal Research Committee. Before transplantation, passage 13 (P13) NPs were examined for nestin, NeuN, and MBP reactivity. Nestin+ NeuN– MBP– NPs were subjected to trypsinization and harvested. Cells were washed twice with PBS and stained with the red fluorescent dye PKH26 according to the manufacturer’s instructions (PKH26 red fluorescent cell linker kit, PKH26-GL; Sigma, St. Louis, http://www.sigmaaldrich.com). Incubations were performed in media containing 2 µM PKH26 for 4.5 minutes at 25°C with 5 x 106 NPs per ml. One injection (1.5 µl) of a 7.5 x 104-cell-per-µl suspension was made into each ventricle. Cell suspensions were delivered at 1 µl per minute using a 5-µl injection needle. The needle was left in situ for 3 minutes postinjection before being slowly removed. The following stereotaxic coordinates were used: anterior-posterior –1.40 mm, medial-lateral 2 mm, and dorsal-ventral –3.8 mm. L32 NSCs, PKH26-labeled L7 oligodendrocyte restricted precursor cells, and nonviable PKH26-labeled P13 NPs were also transplanted. PKH26-labeled (P13) NPs were rendered nonviable by direct submersion in liquid nitrogen and thawing three times in the absence of cryoprotectant. Cell membrane integrity in 100% of cells was destroyed as judged by Trypan Blue staining. However, the cells’ morphologies remained intact. Grafted rats were immunosuppressed by daily injection of cyclosporine A (10 mg/kg, intraperitoneal; Sigma) for the duration of the experiment. In the second, fourth, sixth, and eighth week after transplantation, animals were sacrificed, and the brains were recovered and frozen immediately on dry ice. The entire brain was processed to produce 5-µm-thick cryosections for fluorescence analysis with a confocal laser scanning system, and sections were fixed with 4% paraformaldehyde for further analysis by immunocytochemistry. Based on the original sites of injection, cell migration in brain sections was measured with a microscale. Every 10th section was placed on a glass slide and examined for PKH26+ cells [17]. The proportion of PKH26-labeled cells stained with lineage-specific phenotype markers and a nuclear label (Hoechst 33342) was determined by confocal microscopy. Eight hundred or more cells were scored for each marker (MAP2, GFAP, and MBP) in six animals at the eighth week.

Statistical Analysis
The results are expressed as means ± SEMs. Statistical analysis was performed using the least significant difference test with statistical significance defined as p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
rESC Differentiation into NPs
rESCs, propagated on irradiated MESF [15], were first induced to differentiate spontaneously into EBs by removal from the feeder cells. Day-9 EBs were then cultured under six conditions as described previously. Small-elongated cells were found in differentiating EBs in the G5, HGF+G5, and bFGF groups on day 2 of culture, and in the HGF and S+HGF+G5 groups on day 3–4. In HGF, G5, and HGF+G5 groups, cells were present in both the center and at the periphery of the EBs on day 8 of culture (Figs. 1B–1DGo). In the NDCM control group, after 6 days’ culture, a few small-elongated cells (putative NPs) appeared in the center of the EBs (Fig. 1EGo), with markedly different frequencies dependent on the individual EB (results not shown). Immunostaining showed that small-elongated cells expressed nestin, as described previously [6]. The cell apoptosis rates in the HGF, S+HGF+G5, and HGF+G5 groups were lower than those in the bFGF or G5 groups (results not shown). The rate at which nestin+ NPs appeared under various culture conditions was determined based on an evaluation of 8,500 to 9,000 cells of the total differentiated population in five replicates (Fig. 1AGo). In the control NDCM group, only a few (15% ± 12%) NPs were obtained, which was significantly less (p < .05) than that recovered in the HGF, G5 supplement, and bFGF groups (65% ± 8.3%, 69% ± 14%, and 68.5% ± 7.2%, respectively). In contrast, 88.3% ± 8.1% of NPs were found in the presence of HGF+G5, a statistically significant increase over all other groups (p < .05). Addition of 10% serum to the HGF+G5 group obliterated this improvement (67% ± 12.5%) (p < .05), consistent with the concept previously reported that serum inhibits neural differentiation [2] or that serum chelates a critical component in the medium. There were no differences in the recovery of NPs when the ESCs were first grown on MESF versus on mouse embryonic fibroblast (results not shown).



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  		Figure 1. NP populations from rhesus monkey ESCs under different culture conditions. (A): The rate at which nestin+ NPs in the total differentiated population appeared under various culture conditions was determined with Hoechst 33342 and nestin in five replicates. The results are expressed as means ± SEMs. Statistical analysis was performed using the LSD test, with statistical significance defined as p < .05. The letters (a–c) above the bars represent significant differences between groups (p < .05). (B–D): Small-elongated cells (arrows) appeared at the periphery of plated EBs on day 8 of culture in NDCM medium containing HGF and/or G5: (B) HGF group, (C) G5 group, and (D) HGF+G5 group. (E): A few small-elongated cells appeared in the center of differentiating EBs in the NDCM control group on day 6. (F): Isolated NPs forming spherical structures during in vitro suspension culture. (G): Isolated NPs congregating to form cord-like structures during monolayer culture. (H): Isolated NPs congregating and forming crest-like structures during monolayer culture. Bar scales = 100 µm. Abbreviations: bFGF, basic fibroblast growth factor; EB, embryoid body; ESC, embryonic stem cell; HGF, hepatocyte growth factor; LSD, least significant difference; NDCM, neural differentiation culture media; NP, neural progenitor; S, serum.

 
To further enrich NPs, small-elongated cells from the HGF+G5 group were subjected to trypsin digestion, resulting in purified small-elongated cells in which 98% ± 1.2% were nestin positive based on 7,890 cells examined in four replicates (Figs. 2A, 2BGo). These isolated NPs could be expanded as free-floating neurospheres in suspension culture (Fig. 1FGo). In monolayer culture, some NPs congregated and formed cord-like (Fig. 1GGo) or neural crest–like structures (Fig. 1HGo).



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  		Figure 2. Immunocytochemical characterization of undifferentiated and differentiated NP cells from the HGF+G5 group. (A, B): Nuclear staining and nestin expression, respectively; most (98% ± 1.2%) isolated, small-elongated cells were nestin+. (C): Phase-contrast micrograph of a neurosphere obtained after 4 months of in vitro proliferation of purified NPs. (D): Nestin staining of a neurosphere after in vitro proliferation for 4 months. (E–L): NP differentiation in vitro into neural lineages after the withdrawal of HGF and G5 for 3–4 weeks. Immunocytochemical staining for (E) NeuN, as a mature neuronal marker; (F) synaptophysin, as a mature neuronal marker; (G) merger of (E, F); (H) MAP2, as a neuronal marker; (I) serotonin, as a neuron transmitter; (J) O4, as an oligodendrocyte marker; (K) MBP, as an mature oligodendrocyte marker; and (L) GFAP, as an astrocyte maker. Blue: Hoechst 33342–labeled nuclei. Bar scales = (A– J) 100 µm, (K, L) 50 µm. Abbreviations: GFAP, glial fibrillary acidic protein; HGF, hepatocyte growth factor; MAP2, microtubule-associated protein 2; MBP, myelin basic protein; NeuN, neuronal nuclear antigen; NP, neural progenitor.

 
To evaluate the effect of long-term culture in expansion medium, neurospheres maintained for 4 months were fixed and analyzed for the expression of nestin (Figs. 2C, 2DGo). A high proportion of the cells were immunoreactive to the nestin antibody (96% ± 1.6% of 4,500 cells, n = 4). The 36 population doublings of NPs were received over this 4-month culture.

In Vitro Neural Differentiation
NP neurospheres produced in the presence of HGF+G5 could differentiate into derivatives of all three neural lineages in vitro (neurons, astrocytes, and oligodendrocytes) after withdrawal of the HGF and G5 supplement and plating on laminin or gelatin substrates. In a few days, individual cells and numerous processes grew out of the spheres. After 2–3 weeks, the cells that migrated out and formed a monolayer expressed structural markers of neurons such as NeuN (Figs. 2E, 2GGo), synaptophysin (Figs. 2F, 2GGo), and MAP2 (Fig. 2HGo). A few neuronal cells expressed the neurotransmitter serotonin (Fig. 2IGo). The oligodendrocyte markers, O4+ and MBP+, were observed 4 weeks after HGF and G5 withdrawal (Figs. 2J, 2KGo). GFAP+ astrocytes were rarely found in the first week after HGF and G5 withdrawal; however, they became more frequent after 3 weeks (Fig. 2LGo). NPs derived by monolayer culture could also differentiate into all three neural lineages as described above after HGF and G5 withdrawal.

To determine whether rESC-derived individual NPs that had been cultured in HGF+G5 for 6 weeks were multipotent, we sub-cloned NPs from single cell suspensions. This was accomplished by the micromanipulation under phase-contrast optics with a single cell transferred into individual drops for plating. After 2–3 weeks, 51.5% (53/103) of the drops of inoculated cells contained a colony of cells, and 44 clonal lines were subsequently established. The overall efficiency of subcloning was 42.7%. Several clonal lines were then selected and induced to differentiate for 3 weeks according to the above protocols and stained with anti-MAP2, anti-GFAP, and anti-MBP antibodies (three replicates from three continuous passages). Daughter cells of the L41 line were positive for only the astrocyte marker GFAP (Fig. 3AGo), whereas both of the L20 and L7 lines were positive for only the oligodendrocyte marker MBP (Fig. 3BGo). Clonally derived NSC lines L32, L8, and L22 gave rise to neurons, astrocytes, and oligodendrocytes as evidenced by ICC staining (Fig. 3CGo). The L17 line gave rise to cells expressing both astrocyte and oligodendrocyte markers (Fig. 3DGo). Additionally, we found that L32, L8, and L22 lines differentiated into relatively more astrocytes and oligodendrocytes, whereas the L17 line gave rise to more oligodendrocytes with increasing time of culture in HGF+G5.



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  		Figure 3. Clonally derived monkey neural progenitor lines with unique differentiating abilities. Several lines were induced to differentiate for 3 weeks in the absence of HGF and G5 before staining with anti-MAP2, anti-GFAP, or anti-MBP antibodies. (A): L41 line differentiated into astrocytes only: GFAP+, MBP–, and MAP2–. (B): Both L7 and L20 lines differentiated into oligodendrocytes only: MBP+, GFAP–, and MAP2–. (C): L32, L8, and L22 lines stained positive for neurons, astrocytes, and oligodendrocytes: MAP2+, GFAP+, and MBP+. (D): L17 line showed astrocytes and oligodendrocytes: GFAP+, MBP+, and MAP2–. Blue, Hoechst 33342–labeled nuclei. Bar scales = 50 µm. The arrow denotes MBP-positive cell. Abbreviations: GFAP, glial fibrillary acidic protein; HGF, hepatocyte growth factor; MAP2, microtubule-associated protein 2; MBP, myelin basic protein.

 
Integration and Differentiation in Adult Host Brain
PKH26, a red fluorescent dye, is incorporated into lipid regions of the cell membrane and has been found to be useful for in vitro and in vivo cell labeling, in vitro cell proliferation studies, and in vitro and in vivo cell-tracking applications [18, 19]. Beerheide et al. reported that PKH26-positive cells were examined at 21 days after PKH26-labeled human cord blood somatic stem cells were transplanted into livers of severe combined immunodeficient mice [20]. We tested the stability of the PKH26 dye in NPs by culturing PKH26-labeled cells in vitro for 20 days. During this period, NPs underwent eight population doublings, and 100% of the cells examined displayed PKH26 fluorescence based on an evaluation of 500 cells with confocal microscopy. To further validate the use of PKH26-marked cells in transplantation studies, two control cell populations, clonally derived L7 cells and nonviable, labeled cells, were transplanted into rat brains. The results showed that the PKH26 dye did not migrate from monkey to rat cells (see below) and that viability was required for survival of PKH26+ cells, excluding the possibility that the effects of transplantation treatment were mediated by PKH26 dye (Figs. 4F, 4GGo, 5F, 5GGo).



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  		Figure 4. In vivo integration and migration of monkey neural progenitors (NPs) and clonally derived L32 and L7 lines at 2, 4, 6, and 8 weeks after intracerebroventricular transplantation in adult rats. These figures represent results with nonsubcloned NPs; however, similar findings were obtained for the L32 and L7 clonal lines. NPs are shown (A, B) at the second week, (C) the fourth week, (D, E) the sixth week, and (H) the eighth week. Red cells are donor cells labeled with PKH26. (F, G): PKH26+ cells were not observed in the ventricular wall and parenchyma of control animals that received nonviable, PKH26-labeled NPs (fourth week). Bar scales = 100 µm. White arrows indicate the original injected sites. Black arrows indicate periventricular areas or cortex areas. Encircled areas indicate the size of the engraftment based on observations at the second week (A).

 


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  		Figure 5. In vivo differentiation of rhesus monkey embryonic stem cell–derived NPs and clonally derived, oligodendrocyte-restricted precursor cell (L7) line in the adult rat brain. (A–F): Results for nonsubcloned NPs; however, similar findings were obtained for the subcloned L32 line. (A–C): Cells in the periventricular areas (see labeled areas in Fig. 4Go), whereas (D, E) show cells that have migrated into cortex areas (see labeled areas in Fig 4Go). (A): GFAP (green), astrocyte; (B) MAP2 (green), neuron; (C) MBP (green), oligodendrocyte; (D) oligodendrocyte (MBP) differentiation of NPs migrated to cortex, where the MBP+ proportion was lower than that in periventricular area (C); (E) MAP2+ neuron differentiation of NPs migrated to cortex, where the MAP2+ proportion was higher than that in periventricular area (B). Clone L7 oligodendrocyte restricted precursor cells differentiated only into oligodendrocytes (MBP) (F) and not into neurons (MAP2) (G). Red, PKH26-labeled donor cells; blue, Hoechst 33342–labeled nuclei; yellow, colabeled green (specific fluorescein isothiocyanate–labeled antibody) and red (PKH26) cells (arrow). Bar scales = 50 µm. The insets in (A–C) and (F, G) were higher magnifications of the areas defined by the outlined boxes; bar scales = 20 µm. Abbreviations: GFAP, glial fibrillary acidic protein; HGF, hepatocyte growth factor; MAP2, microtubule-associated protein 2; MBP, myelin basic protein; NP, neural progenitor.

 
After injection of disaggregated PKH26-labeled NPs, there were no behavioral abnormities observed in any of the recipient rats. Fluorescence, histological, and immunochemical evaluation of serial brain sections was performed in animals sacrificed 2, 4, 6, and 8 weeks after transplantation. Numerous PKH26+ cells were found in 22 of the 24 recipients, and successful engraftment was documented with donor cells from P13 NPs and from clones L32 and L7 (Figs. 4A–4E, 4HGo). In two recipients, one at the sixth and the other at the eighth week postinjection, no PKH26+ cells were detected. PKH26+ cells were not observed at the fourth week in the ventricular wall and parenchyma of five control animals that received only nonviable, PKH26-labeled NPs (Figs.4F, 4GGo). Additionally, no teratoma formation was observed in any recipient.

Brains examined in the second week after NP transplantation, that is, PKH26+ NPs, exhibited clusters of donor cells lining the ventricular wall (Figs. 4A, 4BGo). In the fourth to eighth weeks after transplantation, rhesus monkey NP cells left grafted sites and migrated in large numbers, as individual cells or clusters, along the ventricular wall or into the host brain parenchyma (Figs. 4C–4E, 4HGo). The distances that the cells migrated lining the ventricular wall in the second, fourth, sixth, and eighth week, were 100–200 µm, 200–400 µm, 500–700 µm, and 800–1,200 µm, respectively, or approximately 75 µm per week. The distances that cells migrated into brain parenchyma (cerebral cortex) in a radial fashion by the second, fourth, sixth, and eighth week were 100–150 µm, 180–230 µm, 250–300 µm, and 260–320 µm, respectively. Differentiation in vivo into all three neural lineages was demonstrated in triple-labeling experiments with PKH26, Hoechst 33342, and antineural cell type-specific antibodies. The percentage of glial phenotypes was higher than that of neuronal phenotypes in the periventricular areas (Figs. 5A–5CGo). Transplanted cells that differentiated into astrocytes (43% ± 4.6%) and oligodendrocytes (32% ± 5.8%) were also detected by GFAP (Fig. 5AGo) and MBP staining (Fig. 5CGo), respectively. Differentiation into neurons (24% ± 6.2%) in vivo was demonstrated by MAP2 expression (Fig. 5BGo). Oligodendrocyte differentiation rates in the cortex (19% ± 3.2%) were lower than those in the ventricular wall (32% ± 5.8%) (Figs. 5C, 5DGo). In contrast, MAP2+ neuron differentiation rates in the cortex (47% ± 7.9%) were higher than those in the ventricular wall (24% ± 6.2%) (Figs. 5B, 5EGo). L7 cells differentiated only into MBP+ oligodendrocytes (Fig. 5FGo) and not into neurons or astrocytes (Fig. 5GGo).

The nuclear diameter of monkey NPs in vivo was 5 µm, determined from measurements of 50 PKH26-labeled cells. Thus, a 5-µm section should contain one layer of cell nuclei. By carrying out an unbiased sampling of every 10th section (50 µm) and counting the number of PKH26+ cell nuclei, we established that there was a mean survival of 5.2 x 104 ± 980 cells, and therefore, approximately 25% of transplanted cells survived to 2 months after transplantation. Although there were a few cells with nuclei distributed between sections, we excluded nuclei with a diameter of less than 3 µm. Twenty percent to 35% of all surviving PKH26+ cells remained in the injected areas 2 months after transplantation (circles in Fig. 4Go). The proportion of those that migrated along the periventricular wall or into the cortex was 65%–80% of transplanted monkey cells.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neural differentiation of ESCs in vitro provides new perspectives for studying the cellular and molecular mechanisms of early development and carries the prospect of generating donor cells for neural degenerative disease therapy. However, the clinical application of ESCs in neural disease requires the generation of highly purified donor NPs. Schuldiner et al. demonstrated that HGF induced differentiation of human ESCs into ectodermal phenotypes expressing the brain cell marker NF-H [21]. In the present study, we reported culture systems with HGF and G5 supplement that efficiently induce rESC differentiation into NPs. HGF produced results similar to those obtained with bFGF [7], suggesting that HGF may play an important role in early neurongenesis. Other studies have shown that bFGF and EGF as inducers can direct ESC differentiation into NPs or can be used to expand undifferentiated NPs [5–7, 22–24]. In our work, G5 supplement containing EGF and bFGF induced rESC differentiation into NPs. The use of HGF and G5 combined, however, increased the NP differentiation rate over those previously reported [3, 4, 6, 7, 25]. In the present study, we found that in the presence of HGF and G5, NPs were found not only in the center of EBs as reported by others [6, 7] but also at the periphery. This difference may reflect our unique EB culture conditions. When EBs were exposed to bFGF on day 4 or 9 of culture, 8 days later, 10% or 45% of the peripheral cells were nestin positive, respectively. Based on this finding we found that 9-day-old EBs were the most suitable for NP differentiation in this culture system. The results suggested that growth factors (e.g., HGF, G5 supplement) and stages of EBs could cooperatively affect yield of the NPs, which was consistent with the idea that cell differentiation was closely related with time point of action and kinds of growth factors [26].

Highly purified nestin-positive cell populations have been generated by selective dispase digestion and differential adhesion [6]. In this study, we found that NPs were trypsin-sensitive in contrast to non-NPs (flat cells) and differentiated neural cells. Therefore, trypsin (0.125% in 0.04% EDTA) was used to selectively digest NPs that allowed the recovery of cell populations that were 98% nestin-positive. To test whether this approach could be applied to other cultured groups, we purified with trypsin NPs obtained after HGF or bFGF exposure, and 96% of the isolated small-elongated cells were nestin-positive. Moreover, the culture system (HGF+G5) prolonged NP growth and maintained long-term proliferation. Because in vitro differentiation and in vivo transplantation assays showed that NPs produced in this culture system could differentiate into neurons, astrocytes, and oligodendrocytes, the HGF+G5 combination is an efficient inducer to obtain transplantable NPs derived from rESCs.

To further clarify the multipotential nature of rESC-derived NPs, we subcloned NPs. Subclonal analysis showed that some of the clonally derived NSC lines (such as L32, L22, and L8) gave rise to neurons, astrocytes, and oligodendrocytes, whereas others differentiated only into one (L7, L20, and L41) or two (L17) neural lineages. In vivo analysis also demonstrated that clone L32 was multipotent because NPs from this line integrated into rat brain, migrated, and differentiated into neurons, astrocytes, and oligodendrocytes. The high clone rate also indicated that our culture system adequately supported the proliferation of individual NPs. It remains to be determined, however, whether the isolation of subclones with unique differentiation potential is a function of culturing in HGF/G5 or of selecting and propagating NPs that are fundamentally different. In this regard, it would be interesting to conduct a genome-wide expression analysis of these subclonal lines. In any event, subclonal lines with restricted differentiation potential should be valuable for the production of astrocyte or oligodendrocyte phenotypes.

Previous studies used newborn mice or E17 fetuses to assess the differentiation of NPs in vivo [6, 25, 27, 28]. Here, we used adult rats as recipient animals and obtained similar results. Because we injected nestin+, NeuN–, and MBP– NPs, we conclude that PKH26-positive mature neurons, astrocytes, and oligodendrocytes in the engrafted area arose by differentiation and not from injecting differentiated cells. Transplantation of clonal L7 line and nonviable NPs showed that the label (PKH26) did not move between monkey and rat cells. This outcome is consistent with prior studies using PKH26-labeled cells and suggests that it could be used for in vivo neural cell–tracking applications. After transplantation, approximately 25% of the transplanted cells survived at 2 months, and most surviving cells (65%–80%) migrated along the ventricular wall or in a radial fashion. Because transplanted cells differentiated into all three neural lineages, NPs derived by our culturing system from rESCs may be regarded as potential donor cells for neural disease therapy. Interestingly, we found that the kinds and quantities of differentiated cells derived from NPs closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells that migrated into the cortex were mostly neurons. The clonally derived, oligodendrocyte restricted precursor, L7 line differentiated only into oligodendrocytes and, therefore, was not affected by its niche in the rat brain (Figs. 4F, 4GGo). Moreover, transplantations of P13 NPs and lines L32 and L7 derived from NPs suggested that there was an interplay between the cell microenvironment and intrinsic cell properties in the determination of differentiation fate [29, 30]. Our results were consistent with the fact that (a) some cells types transplanted into striatum became mainly GFAP+ [31, 32], whereas others gave rise to mostly neurons [33, 34]; (b) the same cells transplanted together migrated into different sites and displayed different differentiation profiles [35]; and (c) the same cell types transplanted into different brain regions had different differentiation profiles [29, 31, 32].

The present study describes efficient systems for producing highly enriched NPs from rESCs. However, the possible mechanisms by which HGF and G5 regulate rESC differentiation into NPs are unclear and serve as important questions for further research. The challenge in using ESCs for neural regenerative medicine is to direct the differentiation potential of ESCs or NPs into specific neuronal subtypes of interest. Recently, human and monkey ESCs were induced in vitro to adopt NPs, with subsequent successful differentiation into specific neurons in high purity (e.g., midbrain dopaminergic neurons [35, 36] and spinal motoneurons [37]) in response to specific sets of morphogens. Additionally, specific telencephalic precursors from mouse ESCs were obtained [38]. These achievements, along with the present results, make the goal of using ESC-derived NPs in regenerative medicine a distinct possibility. It is now feasible to evaluate whether the highly purified NPs produced here can differentiate into cells such as sensory neurons, motoneurons, dopaminergic neurons, serotonin neurons, and oligodendrocytes and whether they can form functional connections in vivo. It would also be intriguing to test whether specific-region progenitors, such as telencephalic, hindbrain, fore-brain, and neural crest progenitors, can be obtained from rESCs with further modification of culture conditions.

Finally, a demonstration of long-term physiologic function of transplanted cells in rats could lead to analogous engraftment of NPs into monkeys in an effort to bring ESC-based therapies to clinical fruition.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by research grants from Major State Research Development Program 2004CCA01300, G200016108, and 2001cb510100; The Chinese Academy of Sciences KSCX1-05; Chinese National Science Foundation 30370166; and Yunnan Nature Science Foundation 2001C0009Z.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on January 20, 2005; accepted for publication on April 25, 2005.




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