TRANSLATIONAL AND CLINICAL RESEARCH Neuronal Properties, In Vivo Effects, and Pathology of a Huntington’s Disease Patient-Derived Induced Pluripotent Stem Cells IKSOO JEON,a NAYEON LEE,a JIA-YI LI,b,c IN-HYUN PARK,d,e KYOUNG SUN PARK,f JISOOK MOON,a SUNG HAN SHIM,a CHUNGGAB CHOI,a DA-JEONG CHANG,a JIHYE KWON,a SEUNG-HUN OH,a DONG AH SHIN,g HYUN SOOK KIM,a JEONG TAE DO,a DONG RYUL LEE,a MANHO KIM,h KYUNG-SUN KANG,i GEORGE Q. DALEY,d PATRIK BRUNDIN,b,j JIHWAN SONGa CHA Stem Cell Institute, CHA University, Seoul, Korea; bNeuronal Survival Unit, and cNeural Plasticity and Repair Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden; dDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA; eDepartment of Genetics, Yale University School of Medicine, New Haven, USA; fDivision of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang, Korea; iAdult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Korea; jDepartment of Neurosurgery, Yonsei University College of Medicine, Seoul, Korea; hDepartment of Neurology, Seoul National University, College of Medicine; gCenter for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA a
Key Words. Huntington’s disease Aggregate formation
•
Induced pluripotent stem cells
•
GABAergic neurons
•
Quinolinic acid
•
Behavioral recovery
•
ABSTRACT Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can be used to model different human diseases. They may also serve as sources of transplantable cells that can be used in novel cell therapies. Here, we analyzed neuronal properties of an iPSC line derived from a patient with a juvenile form of Huntington’s disease (HD) carrying 72 CAG repeats (HD-iPSC). Although its initial neural inducing activity was lower than that of human embryonic stem cells, we found that HD-iPSC can give rise to GABAergic striatal neurons, the neuronal cell type that is most susceptible to degeneration in HD. We then transplanted HD-iPSC-derived neural precursors into a rat model of HD with a unilateral excitotoxic striatal lesion and observed a significant behavioral recovery in the grafted rats. Interestingly,
during our in vitro culture and when the grafts were examined at 12 weeks after transplantation, no aggregate formation was detected. However, when the culture was treated with a proteasome inhibitor (MG132) or when the cells engrafted into neonatal brains were analyzed at 33 weeks, there were clear signs of HD pathology. Taken together, these results indicate that, although HD-iPSC carrying 72 CAG repeats can form GABAergic neurons and give rise to functional effects in vivo, without showing an overt HD phenotype, it is highly susceptible to proteasome inhibition and develops HD pathology at later stages of transplantation. These unique features of HD-iPSC will serve as useful tools to study HD pathology and develop novel therapeutics. Stem Cells 2012;30:2054– 2062
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION Huntington’s disease (HD) is a devastating, autosomal-dominant neurodegenerative disorder, caused by abnormal expan-
sion of CAG repeats in the huntingtin gene. People carrying the HD mutation gradually develop personality changes, involuntary movements, weight loss, and eventually dementia. Aggregated huntingtin inclusions appear in the cytoplasm and nucleus. One of the affected neuronal populations is the
Author contributions: I.J. and N.L.: conception and design, collection and assembly of data, and data analysis and interpretation; J.Y.L.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; I.H.P.: conception and design and provision of study material; K.S.P., S.H.S., C.C., D.J.C., J.K., S.H.O., D.A.S., J.T.D., and D.R.L.: collection and assembly of data; J.M.: statistical analysis; H.S.K., M.K., and K.S.K: data analysis and interpretation; G.Q.D.: conception and design and provision of study material; P.B.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; J.S.: conception and design, provision of study material, collection and assembly of data, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript. I.J., N.L., and J.-Y.L. contributed equally to this article. Correspondence: Jihwan Song, D.Phil., CHA Stem Cell Institute, Department of Biomedical Science, CHA University, 606-16 Yeoksam 1-dong, Kangnam-gu, Seoul 135-081, Korea. Telephone: þ82-2-3468-3393; Fax: þ82-2-538-4102; e-mail:
[email protected]; or Patrik Brundin, Ph.D., M.D., Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, BMC A10, 221 84 Lund, Sweden. Telephone: þ46-46-222-0529; Fax: þ46-46-222-0531; e-mail:
[email protected] or
[email protected] Received April 19, C AlphaMed Press 1066-5099/ 2011; accepted for publication May 7, 2012; first published online in STEM CELLS EXPRESS May 24, 2012. V 2012/$30.00/0 doi: 10.1002/stem.1135
STEM CELLS 2012;30:2054–2062 www.StemCells.com
Jeon, Lee, Li et al.
2055
striatal medium spiny projection neuron (MSN), which has largely degenerated in end-stage HD [1]. Clinical trials have been performed using grafts of cells derived from human fetal striatal primordium. Outcomes have been variable with some reports describing no benefit while others have indicated transient clinical improvement with reduced motor dysfunction or slowed disease progression [2–4]. Because human fetal tissue transplants raise ethical issues and inevitably are genetically dissimilar to the recipient with the associated risk of immune rejection, other suitable nonfetal cell source of syngeneic donor tissue would be advantageous. The stem cell strategy has been proposed to restore GABAergic striatal projection neurons into the putamen and caudate and re-establish the degenerating striatopallidal circuit [5, 6]. In this study, we have demonstrated that it is possible to differentiate induced pluripotent stem cells (iPSCs) derived from a HD patient carrying 72 CAG repeats [7] into functional GABAergic projection neurons in vitro. After intrastriatal implantation of HD-iPSCNPC, the grafted animals exhibited a clear functional recovery with no overt signs of pathology in the transplanted cells. However, we found that HD-iPSCs develop HD pathology when they are treated with a proteasome inhibitor or when they are grafted into animal models and examined at later stages of transplantation.
MATERIALS
AND
METHODS
Culture and Neuronal Differentiation of HD-iPSC We cultured and maintained HD-iPSC according to the method described previously [8] and induced neuronal differentiation of HD-iPSC by coculturing the cells with PA6 stromal cells (obtained from Riken Cell Bank, Japan) as described previously [9]. For controls, we used a human embryonic stem cell (ESC) (H9, obtained from WiCell, Madison, WI), a normal iPSC (F5), and an additional HD-iPSC line (HD-iPS2) that was derived simultaneously when HD-iPSC was established. These undifferentiated pluripotent stem cell colonies were mechanically dissected and transferred onto freshly prepared PA6 cells in differentiation medium (DM-PA6) that consists of glasgow minimum essential medium (GMEM) containing 10% KO-SR (knockout serum replacements, Invitrogen, Grand Island, NY), and 4 days later, KO-SR in DM-PA6 was replaced by N2 supplements (Invitrogen). In the following 11–13 days, definitive neural rosette-like structures containing neuroepithelial (NE) cells were formed, which were mechanically detached and transferred onto a nonsticky Petri dish for suspension culture for 6 days to form neurospheres.
Stable Generation of Neural Precursor Cells To make single cells at the neural precursor (NP) stage, we dissociated neurospheres following treatment with Accutase (Chemicon, Billerica, MA) and plated onto polyL-ornithine (PLO; 15 lg/ml, Sigma, St. Louis, MO)/fibronectin (FN; 1 lg/ ml, Sigma)-coated 60 mm2 tissue culture dishes. NP cells (NPCs) were maintained in GMEM supplemented with 1% penicillin, 1% streptomycin, 1% nonessential amino acids, 0.1% b-mercaptoethanol, N2 supplements, and 20 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen).
Differentiation into Mature Neurons To differentiate iPSCs into mature neurons (MNs), we plated neurospheres directly onto PLO/FN-coated dishes in DM medium supplemented with 20 ng/ml brain-derived neurotrophic factor (BDNF, R&D Systems, Minneapolis, MN) in the absence of bFGF. www.StemCells.com
Immunocytochemistry To analyze the marker expression of NP-stage and differentiated neuronal cells, we carried out immunocytochemical analyses using the following primary antibodies: human-specific nuclei (1:200; Chemicon), human-specific mitochondria (1:200; Chemicon), human-specific Nestin (1:200; Chemicon), SOX2 (1:200; Chemicon), type III b-tubulin (Tuj1) (1:500; Chemicon), Musashi (1:500; Chemicon), OTX2 (1:500; Chemicon), BF-1 (1:100; Santa Cruz, CA), GSH-2 (1:200; Santa Cruz), DLX2 (1:250; Chemicon), MAP2 (1:200; Chemicon), NeuN (1:500; Chemicon), Gamma-aminobutyric acid (GABA) (1:5,000; Sigma), glutamic acid decarboxylase 65/67 (GAD65/67) (1:200; Chemicon), DARPP-32 (1:100; Cell Signaling, Danvers, MA), Calbindin (1:250; Chemicon), TH (tyrosine hydroxylase, 1:1,000; Pel-Freez, Rogers, AR), SVP38 (1:200; Sigma), and EM48 (1:50; Chemicon). To detect proliferating or undifferentiated cells in the graft, proliferating cell nuclear antigen (PCNA) (1:50; Santa Cruz) and OCT4 (1:250; Santa Cruz) were also used. Secondary antibodies used were goat anti-mouse IgG-conjugated Alexa-555 (1:200; Molecular Probes, Eugene, OR), goat anti-rabbit IgG-conjugated Alexa-488 (1:200; Molecular Probes), and goat anti-mouse IgM-conjugated Alexa-555 (1:200; Molecular Probes). Staining patterns were examined and photographed using a confocal laser-scanning microscope imaging system (LSM510; Carl Zeiss, Inc., Thornwood, NY).
Fluorescence-Activated Cell Sorting Analysis To quantify the percentage of GABAergic neurons formed within the neuronal population of HD-iPSC, we performed fluorescenceactivated cell sorting (FACS) using the FACS Calibur System (BD Bioscience, San Jose, CA). Data analysis was carried out according to manufacturer’s instructions (BD Bioscience).
Electrophysiological Recordings We transferred the cells that were attached to a glass coverslip into a bath mounted on the stage of an inverted microscope (IX-70, Olympus, Osaka, Japan). The bath ( 0.15 ml) was superfused at 5 ml/minute, and voltage-clamp experiments were performed at room temperature (22 C–25 C). Patch pipettes with a free-tip resistance of approximately 2.5–3 MX were connected to the head stage of a patch-clamp amplifier (Axopatch1D, Axon Instruments, Foster City, CA). pCLAMP software v.9.2 and Digidata-1322A (Axon Instruments) were used to acquire data and apply command pulses. Whole-cell currents were recorded at 10 kHz and were low-pass filtered at 1 kHz, respectively. Current traces were stored and analyzed using Clampfit v.9.2 and Origin v.7.0 (Microcal, Northampton, MA). For comparison of whole-cell currents between cells, we normalized the current amplitudes to the membrane area measured by electrical capacitance. Receptor agonist, GABA, and antagonist, picrotoxin, were tested in whole-cell currents.
Quinolinic Acid-Induced Experimental HD Animal Model and Cell Transplantation We carried out animal experiments in accordance with the CHA University IACUC (Institutional Animal Care and Use Committee; IACUC090012). Adult male Sprague-Dawley rats (Orient, Seoul, Korea) weighing 250–280 g were used. All rats received a stereotaxic unilateral lesion of the striatum via double injections of quinolinic acid (QUIN, Fluka, Milwaukee, WI; 0.3 mol/l 1.5 ll) at the following coordinates: AP þ0.7 mm, ML þ2.5 mm, DV –4.5 mm, and AP þ0.7 mm, ML þ2.5 mm, DV –3.5 mm from the Bregma. At 7 days post-QUIN lesion, we injected a total of 12 rats with 2 ll of HD iPS-derived NP cells (100,000 cells per microliter) at the following coordinates: AP þ0.2 mm, ML þ2.2 mm, and DV
In Vitro and In Vivo Properties of HD Patient-Derived iPSC
2056
–4.0 mm from the Bregma. For comparison, we transplanted QUIN-lesioned rats with 2 ll of suspension medium (GMEM) containing the equal numbers of H9-NPC (n ¼ 8) or F5-NPC (n ¼ 11). In the control group (n ¼ 9), 2 ll of GMEM was injected in parallel. Transplanted animals received cyclosporine A intraperitoneally (10 mg/kg, Sigma) 24 hours before transplantation and daily up to 12 weeks.
Behavioral Tests To determine that the QUIN-induced striatal lesions were extensive and to evaluate whether rats recovered functionally following cell transplantation, we performed stepping test, staircase test, and apomorphine-induced rotation tests 1 day prior to transplantation and every 2 weeks following transplantation as previously described [10–12].
Statistical Analysis We performed statistical analyses on the behavior data using the Statistical Analysis System (Enterprise 4.1; SAS Korea, Seoul, Korea) on a CHA University mainframe computer. Performance measures were analyzed using the PROC MIXED program, a linear mixed models procedure. The dependent variables were mean scores from each behavioral test and independent variables were ‘‘Treatment’’ (H9-, HD-, F5-, and sham groups) and ‘‘Week’’ (0w pretransplantation, 2w, 4w, 6w, 8w, 10w, and 12w). The results are presented as the mean 6 SEM. A p value of
