Transplantable Neural Progenitor Populations Derived from Rhesus Monkey Embryoni - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

a Department of Reproduction and Development, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China;
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b Kunming Primate Research Center, The Chinese Academy of Sciences, Kunming, Yunnan, China;

c Graduate School, The Chinese Academy of Sciences, Beijing, China;% S7 x$ t0 T+ D8 I8 b" @
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d Section of Cognitive Brain Research, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China;* V" S0 X* ^% o/ Q  E1 y# i0 C. |

e Oregon National Primate Research Center, Portland, Oregon, USA;& O- S) @) d& s2 |: b& U

f Institute of Zoology, The Chinese Academy of Sciences, Beijing, China8 x  M( e+ y- Z! K3 m/ X
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Key Words. Rhesus monkey embryonic stem cells ? Neural progenitors ? Hepatocyte growth factor ? G5 supplement ? Differentiation) Z* }4 ?4 J& a3 Z. x
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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@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@ioz.ac.cn

ABSTRACT# p$ r  X$ w+ Z( m  n, y1 v
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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.$ Z! P( ^/ g1 s! w1 ?: @/ x6 q  t) E
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Previous studies have shown that ESCs can be induced to generate primitive neural stem cells (NSCs) in chemically defined low-density culture conditions  or in the presence of stromal cell–derived inducing activity (SDIA) . However, only 0.1%–0.2% of ESCs differentiate into neural progenitors (NPs) , and the biological significance of SDIA for neurogenesis is unclear . 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 . In humans and monkeys, neural precursors have been obtained from ESCs . However, a dramatic decrease in cell number occurs, as the majority of the cells do not survive the culture conditions , albeit in low yields; for example, the percentage of neural precursors detectable in differentiated embryoid bodies (EBs) derived from ESCs was less than 80% . Therefore, defining culture conditions that more efficiently induce ESC differentiation into neural precursors is a precondition for further progress in this field.
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Hepatocyte growth factor (HGF) is a pleiotrophic cytokine that can trigger the proliferation, migration, and differentiation of various cell types . Increasing evidence suggests that HGF and its receptor, c-Met, are expressed in the adult and developing nervous system  and that HGF plays an important role in the nervous system . 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 ., R% c6 Z. F. s! X; r/ |; u
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Here, we used defined medium containing HGF and G5 supplement to induce rhesus monkey ESC (rESC) differentiation into transplantable populations of NPs.7 n8 |* y) B2 A( I: S% y

MATERIALS AND METHODS, `3 S& c, V/ ?. c: I# i
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rESC Differentiation into NPs

rESCs, propagated on irradiated MESF , 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–1D). In the NDCM control group, after 6 days’ culture, a few small-elongated cells (putative NPs) appeared in the center of the EBs (Fig. 1E), with markedly different frequencies dependent on the individual EB (results not shown). Immunostaining showed that small-elongated cells expressed nestin, as described previously . 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. 1A). In the control NDCM group, only a few (15% ± 12%) NPs were obtained, which was significantly less (p

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 " g5 l5 G: P8 V# z) ^' W9 p) E+ s# T

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, 2B). These isolated NPs could be expanded as free-floating neurospheres in suspension culture (Fig. 1F). In monolayer culture, some NPs congregated and formed cord-like (Fig. 1G) or neural crest–like structures (Fig. 1H).- B) Y+ i9 c  e& s0 P4 C; n4 x; ~
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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.
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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, 2D). 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.4 k8 W. F) q( x- d* D: u

In Vitro Neural Differentiation4 I% \8 f2 b; J" o1 _7 w

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, 2G), synaptophysin (Figs. 2F, 2G), and MAP2 (Fig. 2H). A few neuronal cells expressed the neurotransmitter serotonin (Fig. 2I). The oligodendrocyte markers, O4  and MBP , were observed 4 weeks after HGF and G5 withdrawal (Figs. 2J, 2K). GFAP  astrocytes were rarely found in the first week after HGF and G5 withdrawal; however, they became more frequent after 3 weeks (Fig. 2L). NPs derived by monolayer culture could also differentiate into all three neural lineages as described above after HGF and G5 withdrawal.
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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. 3A), whereas both of the L20 and L7 lines were positive for only the oligodendrocyte marker MBP (Fig. 3B). Clonally derived NSC lines L32, L8, and L22 gave rise to neurons, astrocytes, and oligodendrocytes as evidenced by ICC staining (Fig. 3C). The L17 line gave rise to cells expressing both astrocyte and oligodendrocyte markers (Fig. 3D). 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.% {6 G) Z) J! H6 d2 q
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Integration and Differentiation in Adult Host Brain
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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 . 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 . 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, 4G, 5F, 5G).

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).; g5 b( S4 K5 w& r, L2 y' A7 v" E
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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. 4), whereas (D, E) show cells that have migrated into cortex areas (see labeled areas in Fig 4). (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.2 N5 J. N3 Y1 @/ s

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, 4H). 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, 4G). Additionally, no teratoma formation was observed in any recipient.
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Brains examined in the second week after NP transplantation, that is, PKH26  NPs, exhibited clusters of donor cells lining the ventricular wall (Figs. 4A, 4B). 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, 4H). 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–5C). Transplanted cells that differentiated into astrocytes (43% ± 4.6%) and oligodendrocytes (32% ± 5.8%) were also detected by GFAP (Fig. 5A) and MBP staining (Fig. 5C), respectively. Differentiation into neurons (24% ± 6.2%) in vivo was demonstrated by MAP2 expression (Fig. 5B). Oligodendrocyte differentiation rates in the cortex (19% ± 3.2%) were lower than those in the ventricular wall (32% ± 5.8%) (Figs. 5C, 5D). 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, 5E). L7 cells differentiated only into MBP  oligodendrocytes (Fig. 5F) and not into neurons or astrocytes (Fig. 5G).
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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. 4). The proportion of those that migrated along the periventricular wall or into the cortex was 65%–80% of transplanted monkey cells.7 u" O) w- b+ o. L- P

DISCUSSION  G0 Q& t4 L7 z( K
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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.4 I2 r" i" j% ~" k4 C
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DISCLOSURES: C$ e7 O3 c9 b( s4 t
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The authors indicate no potential conflicts of interest.2 c: z4 ^) y; A2 F
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