Transplantation of Human Embryonic Stem Cell–Derived Neural Progenitors Improves Behavioral Deficit in Parkinsonian Rats
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《干细胞学杂志》
a Department of Neurology, Agnes Ginges Center for Human Neurogenetics,
b Hadassah Embryonic Stem Cell Research Center, Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel;
c Monash Institute of Reproduction and Development, Monash University, Australia;
d Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel
Key Words. Parkinson’s disease ? Cell therapy ? Human ES cells
Correspondence: Benjamin E. Reubinoff, M.D., Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Ein-Kerem, P.O.B. 12,000, Jerusalem, Israel 91120. Telephone: 972-2-6778589; Fax: 972-2-6430982; e-mail: reubinof@md2.huji.ac.il
ABSTRACT
Pharmacological treatments of Parkinson’s disease have limited long-term success and are associated with serious motor side effects. Transplantation of dopaminergic (DA) neurons is an alternative potential therapeutic approach, and clinical relief of Parkinsonism has been demonstrated in some patients following implantation of fetal-derived DA neurons . However, limited donor tissue supply, ethical considerations, and complicating dyskinesias are major limitations of this mode of therapy.
Pluripotent human embryonic stem cells (hESCs) , which potentially proliferate indefinitely in culture, may supply unlimited numbers of DA neurons for transplantation. The potential of mouse ES cells to generate functional DA neurons and to correct behavioral deficits after engraftment into Parkinsonian rats has been demonstrated . When low numbers of undifferentiated mouse ES cells were implanted into the rat DA-depleted striatum, the cells proliferated and differentiated into functional DA neurons that reduced Parkinsonism. However, lethal teratomas developed in 20% of the animals . In an alternative approach, highly enriched populations of midbrain neural precursors were developed in vitro from mouse ES cells and then implanted into Parkinsonian rats. The engrafted cells led to the recovery from Parkinsonism, and teratoma tumor formation was not observed . The potential therapeutic effect of engrafted hESCs in a Parkinsonian animal model has not yet been evaluated.
We previously derived highly enriched cultures of neural progenitors (NPs) from hESCs . These NPs may serve as a platform for generating DA neurons to treat Parkinsonism. Here, we have implanted the human NPs into Parkinsonian rats. We demonstrate the long-term survival of the graft, lack of teratoma formation, spontaneous differentiation of a relatively small fraction of the transplanted cells into DA neurons, and a significant partial behavioral improvement in the transplanted animals.
MATERIALS AND METHODS
Development and Characterization of hESC-Derived NPs
Differentiation of hESCs into highly enriched cultures of NPs was accomplished according to our two-step protocol with some modifications . In the first step, hESC colonies (Fig. 1A) were cultured for prolonged periods on feeders in the presence of the bone morphogenetic protein antagonist noggin. Under these culture conditions, in most colonies, the hESCs differentiated almost uniformly into tightly packed small progenitors. In parallel, the colonies acquired a nearly uniform gray opaque appearance under dark field stereomicroscope (Fig. 1B). A detailed characterization of these progenitors and the effect of noggin on the differentiation of hESCs are reported elsewhere . In the second step, clumps of 150 progenitors were isolated from the colonies and further propagated for 6 weeks as spheres (Fig. 1C) in serum-free medium supplemented with mitogens . Prior to transplantation, the phenotype of the cells within the spheres and their potential to develop into midbrain DA neurons was characterized. Indirect immunofluorescence analysis (Figs. 1D–G) demonstrated that >90% of the cells within the spheres expressed NP markers (PSA-NCAM 94.7% ± 2.4%, A2B5 90.7% ± 3.2%, NCAM 91.3% ± 2.2%, nestin 94.7% ± 0.7%). Thus, the spheres were highly enriched for NPs.
Figure 1. Derivation and characterization of spheres. Dark field stereomicroscope images of (A) an undifferentiated hESC colony 1 week after passage; (B) noggin-treated colony at 2 weeks after passage; and (C) hESC-derived spheres. Immunostaining of the progenitors for (D) PSA-NCAM, (E) A2B5, (F) NCAM, and (G) nestin. Double immunolabeling showing differentiated NPs co-expressing ?-tubulin III and (H) TH or (I) serotonin, respectively. Semiquantitative reverse transcription–polymerase chain reaction demonstrating the expression of (J) Oct4, markers along dopaminergic neuron development, forebrain and spinal cord markers within cultures of hESCs, and undifferentiated and differentiated NPs. A ± indicates presence or absence of reverse transcriptase. Space bars: (A, B), 100 μm; (C), 200 μm; (D–I), 50 μm. Abbreviations: hESC, human embryonic stem cell; NP, neural progenitor; PSA-NCAM, polysialic acid neural cell adhesion molecule; TH, tyrosine hydroxylase.
Successful differentiation of the hESC-derived NPs into midbrain DA neurons probably requires the induction of the same key regulatory genes that are expressed by NPs during the development of the midbrain in vivo. These genes include the Otx, Pax2, Pax5, En1, En2, Nurr1, and Lmx1b genes . Markers of DA neurons and regulatory genes in their development were expressed by the NPs, suggesting that they had the developmental potential to differentiate into midbrain DA neurons. Markers of other brain areas were also expressed by the spheres. Oct4, a marker of undifferentiated hESCs, was not detected, suggesting that the spheres did not include undifferentiated cells (Fig. 1J).
We further characterized the phenotype of the NPs following spontaneous differentiation in vitro. Upon withdrawal of mitogens from the medium and plating on laminin, the spheres attached rapidly, and cells migrated out to form a monolayer of differentiated cells. After 7 days of differentiation, the expression of the regulatory genes of midbrain development and markers of DA neurons was upregulated in the differentiated progeny (Fig. 1J). Double-labeling studies showed that 29.0% ± 0.6% of the cells were immunoreactive with anti-?-tubulin III (a neuronal marker), 0.56% ± 0.05% of the cells co-expressed ?-tubulin III and TH (Fig. 1H), and 0.89% ± 0.11% co-expressed ?-tubulin III and serotonin (Fig. 1I). These results suggested that a low percentage (<1%) of the progenitors spontaneously differentiated into putative midbrain or hindbrain neurons.
Survival and Differentiation after Transplantation to Parkinsonian Rats
We next explored the survival, differentiation, and function of the NPs after transplantation into the right striatum of Parkinsonian rats. First, 6-hydroxydopamine was injected into the right substantia nigra to deplete dopaminergic innervation in the ipsilateral striatum. At 3 weeks after formation of the lesion, hESC-derived neural spheres that had been passaged for 6 weeks were grafted into the right striatum of rats that were preselected for pharmacological-induced high rotational activity. The rats were sacrificed for histopathological analysis of the graft 24 hours after transplantation (n = 6 animals), and after behavioral follow-up of 12 weeks (21 sphere- and 17 vehicle-grafted rats).
TH immunohistochemistry showed a rich fiber network in the striatum contralateral to the lesion (Fig. 2A), but there was no staining on the lesioned side (Fig. 2B). The grafts were easily identified in brain sections following H&E staining (Fig. 2C), fluorescent DAPI nuclear counterstaining, and indirect immunofluorescence staining for human-specific markers. At 12 weeks after transplantation, a graft was not identified in two rats, and the brains of three rats were processed for RT-PCR analysis (see below). In each of the remaining 16 animals, two grafts were found, most often as a tubular mass of cells along the needle tract within the striatum. In five animals, one of the two grafts was ectopic and was observed as a round mass in the cortex. An inflammatory process was not observed in the transplanted striata or in the control-depleted striata.
Figure 2. Immunohistochemical characterization of transplanted neural progenitors. Immunofluorescence staining for TH in the (A) nonlesioned and (B) lesioned striatum 18 days after 6-hydroxy-dopamine injection. (C): Hematoxylin and eosin staining of a graft 12 weeks after transplantation. (D): Low power field showing the elongated vertical graft in the striatum decorated with human-specific MITO, surrounded by rat tissue. (E): Human identity of cells was confirmed by staining with an anti-RNP antibody (inset: DAPI nuclear counterstain). (F): Immunoreactivity of cells within the graft with human-specific antinestin. (G–J): Confocal microscopy of immunofluorescent-stained sections at 12 weeks post-transplantation showed some human cells expressing markers of neural precursors and also cells expressing glial markers. Confocal images showing grafted cells expressing (G) human mitochondria and mushashi-1, (H) human-specific N-CAM, (I) human-specific CD44, and (J) human mitochondria and GFAP (nuclei indicated by *). (K): Low power image of brain section double stained with antihuman mitochondria and anti–heavy chain NF with DAPI nuclear counterstain, showing that the majority of the transplant did not stain with the neuronal marker. Some NF+ human cells (inset, arrow) were identified mainly near the interface of the graft. (L): Nuclear co-expression of human RNP and NeuN. (M): Double immunostaining of the graft in the striatum showing TH+ cells and fibers within the human mitochondrial–stained graft, with some outgrowth of TH+ fibers at the periphery of graft, into the human mitochondria–negative rat tissue. (N–R): Generation of dopaminergic cells from the transplants. (N):A cell coexpressing TH and human mitochondria within the graft. Confocal microscopy images confirmed the coexpression of human mitochondria and TH or human mitochondria and DAT (R) within the same cells. The nuclei (located by Nomarsky optics) are indicated by asterisks. (S): Immunostaining of the graft for nestin at 24 hours after transplantation. (T–U): Transplanted cells stop proliferating after transplantation. Multiple PCNA+ cells were found at (T) 24 hours post-transplantation but not at (U) 12 weeks after transplantation. Space bars: (G–J) and (L–U), 10 μm; (A, B, D–F, K), 50 μm; (C), 200 μm. All panels apart from (A, B, S, andT) show stainings of grafts 12 weeks after transplantation. Abbreviations: CC, corpus callosum; DAPI, 4',6'-diamidino-2-phenylindole hydrochloride; DAT, human dopamine transporter; GFAP, glial fibrillary acidic protein; MITO, antimitochondria; N-CAM, neural cell adhesion molecule; NeuN, neuronal nuclei marker; NF, neurofilament; PCNA, proliferating cell nuclear antigen; RNP, ribonuclear protein; Str, striatum; TH, tyrosine hydroxylase;V, brain ventricle.
We used human-specific anti-mitochondria (Fig. 2D) and anti-RNP (Fig. 2E) antibodies to specifically identify human cells in transplanted rat brain sections. At 12 weeks post-transplantation, cells that were immunoreactive with the anti-human mitochondria antibodies were found only along the needle tract and within the striatum, and there was no indication of cell migration to neighboring brain regions.
The number of cells within the grafts was evaluated by counting the number of nuclei within the human mitochondria positively stained grafts in serial sections. At 12 weeks post-transplantation, the average number of human cells per striatum was 294,774 ± 72,401 (n = 8), 73.5% of the initial 400,000 cells that were injected into each animal. Survival of the transplanted cells, as well as their proliferation (see below), could contribute to this figure.
Immunostaining with human-specific and neural markers showed cells within the grafts that maintained the phenotype of undifferentiated NPs and expressed nestin, mushashi-1, and NCAM (Fig. 2F–H), as well as differentiated cells expressing the astroglial progenitor marker CD44, the astrocyte marker GFAP, and the neuronal markers heavy chain NF and NeuN (Fig. 2I–L). Double staining for human mitochondria and TH showed the presence of graft-derived TH+ cells and fibers (Fig. 2M–Q). TH+ fibers were commonly observed within the area of distribution of the grafted cells in the host striatum, with some outgrowth from this area into the host striatum (Fig. 2M). In vehicle-grafted animals, there were no TH+ cells in the ipsilateral striatum.
At 12 weeks post-transplantation, the number of TH+ cells in the human mitochondria–stained areas was counted in serial sections spanning the entire graft in six brains and adjusted to the full volume of the graft. The grafts generated 389 ± 83 TH+ neurons (102–630 TH+ neurons per brain), which were 0.18% ± 0.05% of the total number of cells within the graft.
Cells that were decorated with an antibody directed against human dopamine transporter, a specific marker of DA neurons, were identified within the graft and were undetectable in the striatum of medium-grafted controls (Fig. 2R).
To further confirm the expression of human dopaminergic neuronal markers in the transplanted brains, we performed RT-PCR analysis of striatal samples from vehicle-grafted (n = 2) and NP-grafted (n = 3) rats. Human-specific transcripts of midbrain and DA neuron markers were expressed only in samples from NP-grafted animals (Fig. 3F). Expression of the transcripts was found only on the transplanted side, and not on the nonlesioned side of the same animals (not shown).
Figure 3. Transplantation of human embryonic stem cell–derived NPs improves motor function in Parkinsonian rats. The number of apomorphine- or D-amphetamine–induced rotations was calculated individually for each rat as a percentage of its performance at baseline. For each time point, the value represents the mean ± standard error percentage of rotations. Rotational behavior that was induced by (A) apomorphine, and (B) D-amphetamine decreased significantly in transplanted animals, as compared with baseline and control rats. Both (C) stepping and (D) placing are indicated as the percentage of forelimb stepping adjustments and placing in the lesioned side, as compared with the nonlesioned side. Both tests improved significantly (p = .0012 and .0003, respectively) in transplanted rats, as compared with controls. (E): The total number of TH+ neurons in the graft of individual rats correlated with the extent of reduction of amphetamine-induced rotations at 12 weeks following transplantation (r = –.97; p = .001). (F): Reverse transcription polymerase chain reaction was used to analyze striata samples from sphere-grafted and vehicle-grafted animals for the expression of human-specific transcripts of midbrain and dopaminergic neuron markers. The human-specific transcripts (for En1, En2, TH, L-AADC, and GAPDH) were expressed only by animals that received NPs and were not detected in sham-operated animals. ?-actin primers were not human specific. A ± indicates presence or absence of reverse transcriptase. , controls;, transplanted group. *p < .05, as compared with baseline and controls for the pharmacological tests and with the control group for the nonpharmacological tests. Abbreviations: AADC, acid decarboxylase; GAPDH, glyceraldehyde-3-phosphate; NP, neural progenitors; TH, tyrosine hydroxylase.
Given the potential of ES cells to generate teratomas after transplantation, we evaluated the percentage of proliferating cells within the grafts. At 24 hours post-transplantation, the grafts were heavily stained with the neural precursor cell marker nestin (Fig. 2S). At that time, the majority (64.5% ± 0.9%) of cells within the graft were in a proliferative state, as indicated by their immunoreactivity with anti-PCNA (Fig. 2T) and anti-ki67 (not shown). At 12 weeks, there were very rare PCNA+ cells (0.2% ± 0.05%; Fig. 2U). In addition, serial H&E-stained sections covering the entire brain did not reveal teratomas or any other tumor formation in transplanted rats.
Functional Recovery of Sphere-Grafted Parkinsonian Rats
Pharmacological-induced rotational behavior was measured in rats that were transplanted either with spheres or with medium at 2 weeks (baseline), 4 weeks, 8 weeks, and 12 weeks after engraftment. Two rats in which no graft was found did not exhibit any improvement in motor function and were excluded from the analysis. In transplanted rats (n = 19), apomorphine-induced rotations decreased in the transplanted group from an average of 624 ± 50 times per hour at baseline to 423 ± 36 rotations per hour at 12 weeks (31% decrease, p = .0015; Fig. 3A). The control group of rats (n = 17) rotated 567 ± 41 times per hour at baseline and 571 ± 27 after 12 weeks. Amphetamine-induced rotations decreased in the transplanted group of rats (n = 10) from 607 ± 63 per hour at baseline to 334 ± 41 per hour at 12 weeks (45% decrease, p = .001; Fig. 3B). Amphetamine-induced rotations increased in the control group (n = 10) from 480 ± 66 times per hour to 571 ± 74 rotations per hour at 12 weeks. The total number of TH+ neurons in the engrafted striatum, at 12 weeks after transplantation, was compared with the degree of behavioral improvement (n = 6 animals; Fig. 3E). A significant correlation was found between the number of TH+ neurons and the degree of improvement in amphetamine-induced rotational behavior (r = –.97, p = .001).
Stepping adjustments and forelimb placing are nonpharmacological tests, which may provide a more direct measure of motor deficits that are analogous to those found in human Parkinson’s disease . Stepping and placing were examined at baseline (2 weeks) and at 12 weeks after transplantation. At 2 weeks, the transplanted rats (n = 11) did not make any stepping or placing movements on the lesioned side. At 12 weeks, there was a significant improvement in both nonpharmacological tests and in baseline and control rats (n = 10; Fig. 3C–D).
DISCUSSION
In conclusion, we show for the first time partial functional recovery following transplantation of hESC-derived NPs in an experimental model of Parkinson’s disease. The transplanted NPs were not directed by the host striatum to differentiate into DA neurons; therefore, induction of their differentiation into a DA fate, prior to transplantation, may potentially improve the functional effect that we have observed . Our observations encourage further efforts that may eventually allow the use of hESCs for the treatment of Parkinson’s disease.
ACKNOWLEDGMENTS
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b Hadassah Embryonic Stem Cell Research Center, Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel;
c Monash Institute of Reproduction and Development, Monash University, Australia;
d Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel
Key Words. Parkinson’s disease ? Cell therapy ? Human ES cells
Correspondence: Benjamin E. Reubinoff, M.D., Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Ein-Kerem, P.O.B. 12,000, Jerusalem, Israel 91120. Telephone: 972-2-6778589; Fax: 972-2-6430982; e-mail: reubinof@md2.huji.ac.il
ABSTRACT
Pharmacological treatments of Parkinson’s disease have limited long-term success and are associated with serious motor side effects. Transplantation of dopaminergic (DA) neurons is an alternative potential therapeutic approach, and clinical relief of Parkinsonism has been demonstrated in some patients following implantation of fetal-derived DA neurons . However, limited donor tissue supply, ethical considerations, and complicating dyskinesias are major limitations of this mode of therapy.
Pluripotent human embryonic stem cells (hESCs) , which potentially proliferate indefinitely in culture, may supply unlimited numbers of DA neurons for transplantation. The potential of mouse ES cells to generate functional DA neurons and to correct behavioral deficits after engraftment into Parkinsonian rats has been demonstrated . When low numbers of undifferentiated mouse ES cells were implanted into the rat DA-depleted striatum, the cells proliferated and differentiated into functional DA neurons that reduced Parkinsonism. However, lethal teratomas developed in 20% of the animals . In an alternative approach, highly enriched populations of midbrain neural precursors were developed in vitro from mouse ES cells and then implanted into Parkinsonian rats. The engrafted cells led to the recovery from Parkinsonism, and teratoma tumor formation was not observed . The potential therapeutic effect of engrafted hESCs in a Parkinsonian animal model has not yet been evaluated.
We previously derived highly enriched cultures of neural progenitors (NPs) from hESCs . These NPs may serve as a platform for generating DA neurons to treat Parkinsonism. Here, we have implanted the human NPs into Parkinsonian rats. We demonstrate the long-term survival of the graft, lack of teratoma formation, spontaneous differentiation of a relatively small fraction of the transplanted cells into DA neurons, and a significant partial behavioral improvement in the transplanted animals.
MATERIALS AND METHODS
Development and Characterization of hESC-Derived NPs
Differentiation of hESCs into highly enriched cultures of NPs was accomplished according to our two-step protocol with some modifications . In the first step, hESC colonies (Fig. 1A) were cultured for prolonged periods on feeders in the presence of the bone morphogenetic protein antagonist noggin. Under these culture conditions, in most colonies, the hESCs differentiated almost uniformly into tightly packed small progenitors. In parallel, the colonies acquired a nearly uniform gray opaque appearance under dark field stereomicroscope (Fig. 1B). A detailed characterization of these progenitors and the effect of noggin on the differentiation of hESCs are reported elsewhere . In the second step, clumps of 150 progenitors were isolated from the colonies and further propagated for 6 weeks as spheres (Fig. 1C) in serum-free medium supplemented with mitogens . Prior to transplantation, the phenotype of the cells within the spheres and their potential to develop into midbrain DA neurons was characterized. Indirect immunofluorescence analysis (Figs. 1D–G) demonstrated that >90% of the cells within the spheres expressed NP markers (PSA-NCAM 94.7% ± 2.4%, A2B5 90.7% ± 3.2%, NCAM 91.3% ± 2.2%, nestin 94.7% ± 0.7%). Thus, the spheres were highly enriched for NPs.
Figure 1. Derivation and characterization of spheres. Dark field stereomicroscope images of (A) an undifferentiated hESC colony 1 week after passage; (B) noggin-treated colony at 2 weeks after passage; and (C) hESC-derived spheres. Immunostaining of the progenitors for (D) PSA-NCAM, (E) A2B5, (F) NCAM, and (G) nestin. Double immunolabeling showing differentiated NPs co-expressing ?-tubulin III and (H) TH or (I) serotonin, respectively. Semiquantitative reverse transcription–polymerase chain reaction demonstrating the expression of (J) Oct4, markers along dopaminergic neuron development, forebrain and spinal cord markers within cultures of hESCs, and undifferentiated and differentiated NPs. A ± indicates presence or absence of reverse transcriptase. Space bars: (A, B), 100 μm; (C), 200 μm; (D–I), 50 μm. Abbreviations: hESC, human embryonic stem cell; NP, neural progenitor; PSA-NCAM, polysialic acid neural cell adhesion molecule; TH, tyrosine hydroxylase.
Successful differentiation of the hESC-derived NPs into midbrain DA neurons probably requires the induction of the same key regulatory genes that are expressed by NPs during the development of the midbrain in vivo. These genes include the Otx, Pax2, Pax5, En1, En2, Nurr1, and Lmx1b genes . Markers of DA neurons and regulatory genes in their development were expressed by the NPs, suggesting that they had the developmental potential to differentiate into midbrain DA neurons. Markers of other brain areas were also expressed by the spheres. Oct4, a marker of undifferentiated hESCs, was not detected, suggesting that the spheres did not include undifferentiated cells (Fig. 1J).
We further characterized the phenotype of the NPs following spontaneous differentiation in vitro. Upon withdrawal of mitogens from the medium and plating on laminin, the spheres attached rapidly, and cells migrated out to form a monolayer of differentiated cells. After 7 days of differentiation, the expression of the regulatory genes of midbrain development and markers of DA neurons was upregulated in the differentiated progeny (Fig. 1J). Double-labeling studies showed that 29.0% ± 0.6% of the cells were immunoreactive with anti-?-tubulin III (a neuronal marker), 0.56% ± 0.05% of the cells co-expressed ?-tubulin III and TH (Fig. 1H), and 0.89% ± 0.11% co-expressed ?-tubulin III and serotonin (Fig. 1I). These results suggested that a low percentage (<1%) of the progenitors spontaneously differentiated into putative midbrain or hindbrain neurons.
Survival and Differentiation after Transplantation to Parkinsonian Rats
We next explored the survival, differentiation, and function of the NPs after transplantation into the right striatum of Parkinsonian rats. First, 6-hydroxydopamine was injected into the right substantia nigra to deplete dopaminergic innervation in the ipsilateral striatum. At 3 weeks after formation of the lesion, hESC-derived neural spheres that had been passaged for 6 weeks were grafted into the right striatum of rats that were preselected for pharmacological-induced high rotational activity. The rats were sacrificed for histopathological analysis of the graft 24 hours after transplantation (n = 6 animals), and after behavioral follow-up of 12 weeks (21 sphere- and 17 vehicle-grafted rats).
TH immunohistochemistry showed a rich fiber network in the striatum contralateral to the lesion (Fig. 2A), but there was no staining on the lesioned side (Fig. 2B). The grafts were easily identified in brain sections following H&E staining (Fig. 2C), fluorescent DAPI nuclear counterstaining, and indirect immunofluorescence staining for human-specific markers. At 12 weeks after transplantation, a graft was not identified in two rats, and the brains of three rats were processed for RT-PCR analysis (see below). In each of the remaining 16 animals, two grafts were found, most often as a tubular mass of cells along the needle tract within the striatum. In five animals, one of the two grafts was ectopic and was observed as a round mass in the cortex. An inflammatory process was not observed in the transplanted striata or in the control-depleted striata.
Figure 2. Immunohistochemical characterization of transplanted neural progenitors. Immunofluorescence staining for TH in the (A) nonlesioned and (B) lesioned striatum 18 days after 6-hydroxy-dopamine injection. (C): Hematoxylin and eosin staining of a graft 12 weeks after transplantation. (D): Low power field showing the elongated vertical graft in the striatum decorated with human-specific MITO, surrounded by rat tissue. (E): Human identity of cells was confirmed by staining with an anti-RNP antibody (inset: DAPI nuclear counterstain). (F): Immunoreactivity of cells within the graft with human-specific antinestin. (G–J): Confocal microscopy of immunofluorescent-stained sections at 12 weeks post-transplantation showed some human cells expressing markers of neural precursors and also cells expressing glial markers. Confocal images showing grafted cells expressing (G) human mitochondria and mushashi-1, (H) human-specific N-CAM, (I) human-specific CD44, and (J) human mitochondria and GFAP (nuclei indicated by *). (K): Low power image of brain section double stained with antihuman mitochondria and anti–heavy chain NF with DAPI nuclear counterstain, showing that the majority of the transplant did not stain with the neuronal marker. Some NF+ human cells (inset, arrow) were identified mainly near the interface of the graft. (L): Nuclear co-expression of human RNP and NeuN. (M): Double immunostaining of the graft in the striatum showing TH+ cells and fibers within the human mitochondrial–stained graft, with some outgrowth of TH+ fibers at the periphery of graft, into the human mitochondria–negative rat tissue. (N–R): Generation of dopaminergic cells from the transplants. (N):A cell coexpressing TH and human mitochondria within the graft. Confocal microscopy images confirmed the coexpression of human mitochondria and TH or human mitochondria and DAT (R) within the same cells. The nuclei (located by Nomarsky optics) are indicated by asterisks. (S): Immunostaining of the graft for nestin at 24 hours after transplantation. (T–U): Transplanted cells stop proliferating after transplantation. Multiple PCNA+ cells were found at (T) 24 hours post-transplantation but not at (U) 12 weeks after transplantation. Space bars: (G–J) and (L–U), 10 μm; (A, B, D–F, K), 50 μm; (C), 200 μm. All panels apart from (A, B, S, andT) show stainings of grafts 12 weeks after transplantation. Abbreviations: CC, corpus callosum; DAPI, 4',6'-diamidino-2-phenylindole hydrochloride; DAT, human dopamine transporter; GFAP, glial fibrillary acidic protein; MITO, antimitochondria; N-CAM, neural cell adhesion molecule; NeuN, neuronal nuclei marker; NF, neurofilament; PCNA, proliferating cell nuclear antigen; RNP, ribonuclear protein; Str, striatum; TH, tyrosine hydroxylase;V, brain ventricle.
We used human-specific anti-mitochondria (Fig. 2D) and anti-RNP (Fig. 2E) antibodies to specifically identify human cells in transplanted rat brain sections. At 12 weeks post-transplantation, cells that were immunoreactive with the anti-human mitochondria antibodies were found only along the needle tract and within the striatum, and there was no indication of cell migration to neighboring brain regions.
The number of cells within the grafts was evaluated by counting the number of nuclei within the human mitochondria positively stained grafts in serial sections. At 12 weeks post-transplantation, the average number of human cells per striatum was 294,774 ± 72,401 (n = 8), 73.5% of the initial 400,000 cells that were injected into each animal. Survival of the transplanted cells, as well as their proliferation (see below), could contribute to this figure.
Immunostaining with human-specific and neural markers showed cells within the grafts that maintained the phenotype of undifferentiated NPs and expressed nestin, mushashi-1, and NCAM (Fig. 2F–H), as well as differentiated cells expressing the astroglial progenitor marker CD44, the astrocyte marker GFAP, and the neuronal markers heavy chain NF and NeuN (Fig. 2I–L). Double staining for human mitochondria and TH showed the presence of graft-derived TH+ cells and fibers (Fig. 2M–Q). TH+ fibers were commonly observed within the area of distribution of the grafted cells in the host striatum, with some outgrowth from this area into the host striatum (Fig. 2M). In vehicle-grafted animals, there were no TH+ cells in the ipsilateral striatum.
At 12 weeks post-transplantation, the number of TH+ cells in the human mitochondria–stained areas was counted in serial sections spanning the entire graft in six brains and adjusted to the full volume of the graft. The grafts generated 389 ± 83 TH+ neurons (102–630 TH+ neurons per brain), which were 0.18% ± 0.05% of the total number of cells within the graft.
Cells that were decorated with an antibody directed against human dopamine transporter, a specific marker of DA neurons, were identified within the graft and were undetectable in the striatum of medium-grafted controls (Fig. 2R).
To further confirm the expression of human dopaminergic neuronal markers in the transplanted brains, we performed RT-PCR analysis of striatal samples from vehicle-grafted (n = 2) and NP-grafted (n = 3) rats. Human-specific transcripts of midbrain and DA neuron markers were expressed only in samples from NP-grafted animals (Fig. 3F). Expression of the transcripts was found only on the transplanted side, and not on the nonlesioned side of the same animals (not shown).
Figure 3. Transplantation of human embryonic stem cell–derived NPs improves motor function in Parkinsonian rats. The number of apomorphine- or D-amphetamine–induced rotations was calculated individually for each rat as a percentage of its performance at baseline. For each time point, the value represents the mean ± standard error percentage of rotations. Rotational behavior that was induced by (A) apomorphine, and (B) D-amphetamine decreased significantly in transplanted animals, as compared with baseline and control rats. Both (C) stepping and (D) placing are indicated as the percentage of forelimb stepping adjustments and placing in the lesioned side, as compared with the nonlesioned side. Both tests improved significantly (p = .0012 and .0003, respectively) in transplanted rats, as compared with controls. (E): The total number of TH+ neurons in the graft of individual rats correlated with the extent of reduction of amphetamine-induced rotations at 12 weeks following transplantation (r = –.97; p = .001). (F): Reverse transcription polymerase chain reaction was used to analyze striata samples from sphere-grafted and vehicle-grafted animals for the expression of human-specific transcripts of midbrain and dopaminergic neuron markers. The human-specific transcripts (for En1, En2, TH, L-AADC, and GAPDH) were expressed only by animals that received NPs and were not detected in sham-operated animals. ?-actin primers were not human specific. A ± indicates presence or absence of reverse transcriptase. , controls;, transplanted group. *p < .05, as compared with baseline and controls for the pharmacological tests and with the control group for the nonpharmacological tests. Abbreviations: AADC, acid decarboxylase; GAPDH, glyceraldehyde-3-phosphate; NP, neural progenitors; TH, tyrosine hydroxylase.
Given the potential of ES cells to generate teratomas after transplantation, we evaluated the percentage of proliferating cells within the grafts. At 24 hours post-transplantation, the grafts were heavily stained with the neural precursor cell marker nestin (Fig. 2S). At that time, the majority (64.5% ± 0.9%) of cells within the graft were in a proliferative state, as indicated by their immunoreactivity with anti-PCNA (Fig. 2T) and anti-ki67 (not shown). At 12 weeks, there were very rare PCNA+ cells (0.2% ± 0.05%; Fig. 2U). In addition, serial H&E-stained sections covering the entire brain did not reveal teratomas or any other tumor formation in transplanted rats.
Functional Recovery of Sphere-Grafted Parkinsonian Rats
Pharmacological-induced rotational behavior was measured in rats that were transplanted either with spheres or with medium at 2 weeks (baseline), 4 weeks, 8 weeks, and 12 weeks after engraftment. Two rats in which no graft was found did not exhibit any improvement in motor function and were excluded from the analysis. In transplanted rats (n = 19), apomorphine-induced rotations decreased in the transplanted group from an average of 624 ± 50 times per hour at baseline to 423 ± 36 rotations per hour at 12 weeks (31% decrease, p = .0015; Fig. 3A). The control group of rats (n = 17) rotated 567 ± 41 times per hour at baseline and 571 ± 27 after 12 weeks. Amphetamine-induced rotations decreased in the transplanted group of rats (n = 10) from 607 ± 63 per hour at baseline to 334 ± 41 per hour at 12 weeks (45% decrease, p = .001; Fig. 3B). Amphetamine-induced rotations increased in the control group (n = 10) from 480 ± 66 times per hour to 571 ± 74 rotations per hour at 12 weeks. The total number of TH+ neurons in the engrafted striatum, at 12 weeks after transplantation, was compared with the degree of behavioral improvement (n = 6 animals; Fig. 3E). A significant correlation was found between the number of TH+ neurons and the degree of improvement in amphetamine-induced rotational behavior (r = –.97, p = .001).
Stepping adjustments and forelimb placing are nonpharmacological tests, which may provide a more direct measure of motor deficits that are analogous to those found in human Parkinson’s disease . Stepping and placing were examined at baseline (2 weeks) and at 12 weeks after transplantation. At 2 weeks, the transplanted rats (n = 11) did not make any stepping or placing movements on the lesioned side. At 12 weeks, there was a significant improvement in both nonpharmacological tests and in baseline and control rats (n = 10; Fig. 3C–D).
DISCUSSION
In conclusion, we show for the first time partial functional recovery following transplantation of hESC-derived NPs in an experimental model of Parkinson’s disease. The transplanted NPs were not directed by the host striatum to differentiate into DA neurons; therefore, induction of their differentiation into a DA fate, prior to transplantation, may potentially improve the functional effect that we have observed . Our observations encourage further efforts that may eventually allow the use of hESCs for the treatment of Parkinson’s disease.
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