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Maturation of human iPS cell-derived dopamine neuron precursors in alginate–Ca2 + hydrogel
Biochimica et Biophysica Acta 1850 (2015) 1669–1675
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Maturation of human iPS cell-derived dopamine neuron precursors in alginate–Ca2 + hydrogel Mitsue Komatsu, Shuhei Konagaya, Edgar Y. Egawa, Hiroo Iwata ⁎ Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 14 April 2015 Accepted 27 April 2015 Available online 4 May 2015 Keywords: iPS cell Dopamine Neuron precursor Alginate Microencapsulation
a b s t r a c t Background: Pluripotent stem cells (embryonic stem/induced pluripotent stem cells) have been widely studied as a potential cell source for cell transplantation therapy of Parkinson's disease. However, some difficulties remain to be overcome. These include the need to prepare a large number of dopamine (DA) neurons for clinical use and to culture the cells for a long period to allow their functional maturation and the removal of undifferentiated cells. Methods: In this study, aggregates of DA neuron precursors were enclosed in alginate–Ca2+ microbeads, and the encapsulated aggregates were cultured for 25 days to induce cell maturation. Results: More than 60% of cells in the aggregates differentiated into tyrosine hydroxylase-positive DA neurons. The aggregates could release DA at the same level as aggregates maintained on culture dishes without encapsulation. In addition, by exposure to a citrate solution, the alginate–Ca2+ gel layer could be easily removed from aggregates without damaging the DA neurons. When the aggregates were transplanted into rat brain, viable cells were found in the graft at one week post-transplantation, with cells extending neurites into the host tissue. Conclusions: Cell aggregates encapsulated in alginate–Ca2+ beads successfully differentiated into mature DA neurons. General significance: The alginate–Ca2+ microbead is suitable for maintaining DA precursor aggregates for a long period to allow their functional maturation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pluripotent stem cells, such as embryonic stem (ES) and induced pluripotent stem (iPS) cells, can be expanded without limit and induced to differentiate into multiple functional cell types and have been considered as potential cell sources for cell transplantation therapy of Parkinson's disease (PD) [1]. Protocols for differentiation of pluripotent cells into dopamine (DA) neurons have been widely studied [2–4], and differentiated DA neurons from ES cells or iPS cells have been transplanted into PD model animals to demonstrate efficacy [5, 6]. Although DA neurons derived from human pluripotent stem cells are being considered as treatment for PD patients in several countries, some difficulties remain to be overcome. One of these is that huge numbers of DA neurons must be prepared for clinical application because the human body is about 3000 times larger than that of a mouse. Another problem requiring careful consideration with transplantation of iPSderived cells is tumor formation [8, 9] because undifferentiated cells contaminating grafts might form tumors. It has been claimed that the risk of tumor formation can be reduced by maturing cells for a long period of culture in vitro before transplantation [10, 11].
⁎ Corresponding author. Tel.: +81 75 751 4119; fax: +81 75 751 4646. E-mail address: [email protected] (H. Iwata).
http://dx.doi.org/10.1016/j.bbagen.2015.04.011 0304-4165/© 2015 Elsevier B.V. All rights reserved.
In our previous studies [7], we examined microencapsulation of DA neuron precursors in agarose beads as a way to overcome these two difficulties. In that work [7], DA neuron precursors effectively matured by long-term culture in agarose beads, and the cells released DA for more than 40 days. In addition, microbeads containing cells could be cryopreserved. Although agarose microencapsulation provides a good supporting environment for the preparation and storage of DA neurons, the tedious process of removal of agarose microbeads by hand is required before transplantation to allow their direct interaction with host tissue. In the current study, we evaluated alginate–Ca2+ hydrogel for microencapsulation of DA neuron precursors. An alginate solution turns into gel when it is exposed to a solution of a divalent metal ion such as Ca2 + and Ba2 +. Alginate–Ca2 + hydrogels have been widely examined for preparation of a bioartificial pancreas, specifically microencapsulated islets of Langerhans [12, 13]. The gel can be returned to solution by the addition of chelating agents, such as citric acid or ethylenediamine-tetraacetic acid (EDTA). It has been reported that cells are easily encapsulated in alginate–Ca2+ gel and can be retrieved undamaged from the capsule under moderate conditions [14]. Alginate–Ca2+ thus seems to be a suitable material for microencapsulation of DA neuron precursors and their long-term culture. Sometimes, a minor difference in cell culture medium, however, drastically affects cell fate, such as survival and direction of differentiation. In this study,
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aggregates of DA neuron precursors were enclosed in alginate–Ca2 + microbeads and cultured for a long period. In an alginate–Ca2 + microbead, cells are exposed to dense anionic functional groups, carboxyl side groups of alginate, and low Ca2+ concentration. Their effects on the fate of DA neuron precursors, number of tyrosine hydroxylase (TH)-positive cells, and DA release function were examined.
suspension was dropped through a 25-G hypodermic needle into 100 mM CaCl2 solution containing 2 mM KCl and 1 mM HEPES. After 10 min for complete gelation, the alginate microbeads were washed twice with physiological saline. The encapsulated cell aggregates were cultured in the basal medium for an additional 25 days for maturation into DA neurons. Culture medium was replaced with fresh medium every 2 or 3 days.
2. Materials and methods 2.1. Human iPS (hiPS) cell culture and differentiation into DA neurons Two lines of hiPS cells (201B7 [15] and 253G1 [16], RIKEN Cell Bank, Ibaraki, Japan) were used in this study. Undifferentiated hiPS cells were maintained on an SNL 76/7 (ECACC, Salisbury, UK) feeder layer as previously described [17]. HiPS cells were differentiated into the DA neuronal lineage following the protocol shown in Scheme 1 [7]. Briefly, undifferentiated hiPS cells were seeded on Matrigel (BD Bioscience, San Jose, CA, USA) and cultured in medium containing several cytokines and small molecules. Differentiation medium was changed daily, and on day 18, subpopulations of cells were used for microencapsulation. The remaining cells were subcultured on a laminin/poly-L-ornithine (LA/ PLO)-coated dish for additional days. 2.2. Formation of cell aggregates On day 18, cells were detached from culture dishes by treatment with Accumax (Innovative Cell Technologies, Inc., San Diego, CA, USA) for 3 min. The cells were suspended in DMEM/F12 supplemented with 2.5 mM GlutaMAX, 2% B27 supplement (Invitrogen), 10 ng/mL brainderived neurotrophic growth factor (Wako Pure Chemical Industries, Osaka, Japan), 10 ng/mL glial cell line-derived neurotrophic growth factor (Wako), 0.2 mM ascorbic acid (Nacalai Tesque), 0.5 mM dibutyryl cyclic AMP (dcAMP, Nacalai Tesque), 1 ng/mL transforming growth factor-β3 (TGF-β3, R&D Systems), 100 U/mL penicillin, and 100 μg/mL streptomycin. This medium is referred to hereafter as the basal medium. U-bottom 96-well plates were pre-coated with 2% Pluronic® F127 overnight to inhibit cell adhesion. After plating, the cells were washed five times with phosphate buffered saline (PBS), applied to wells at a density of 6000 cells/well, and centrifuged at 1000 rpm for 3 min. The cells were cultured in an incubator at 37 °C in 5% CO2 atmosphere for 5 days to prepare cell aggregates. 2.3. Encapsulation Cell aggregates were enclosed in alginate–Ca2 + beads. Approximately 1000 cell aggregates were mixed with 2 mL of 1% alginate (Mw; 3 × 105, Nacalai Tesque) in Krebs–Ringer/HEPES solution. The
Scheme 1. Experimental protocol. HiPS cells were differentiated into DA neurons in a series of media supplemented with different inhibitors and cytokines. DA precursor cells were collected from the dish at day 18, and 6000 cells were applied to each well of 96well U-bottom plates that were pretreated with Pluronic® 127F. The cells were cultured for an additional 5 days to form cell aggregates, which were then encapsulated in alginate beads on day 23. Encapsulated cell aggregates were cultured in the same medium type as that used for aggregate formation. On day 48, alginate hydrogel was dissolved by exposure to a citrate buffer solution, and cell aggregates were collected.
2.4. Decapsulation On day 48, alginate–Ca2+ beads were dissolved to collect cell aggregates. After being washed with saline solution three times, the alginate– Ca2+ beads were treated with 50 mM citrate buffer solution in physiological saline solution for 15 min at 37 °C. The cell aggregates were washed two times with PBS and then seeded onto a LM/PLO-coated dish and cultured for one week. The cell aggregates that adhered on the LM/PLO-coated dish were subjected to immunofluorescence examination. 2.5. DA secretion Six hundred decapsulated cell aggregates were washed twice with PBS supplemented with 0.33 mM Mg2+ and 0.9 mM Ca2+. The cells were incubated for 30 min in 56 mM KCl/Hanks balanced salt solution supplemented with 0.33 mM Mg2+ and 0.9 mM Ca2+ to induce depolarization of the cell membrane. A total of 300 μL of the supernatant was collected into a test tube, and 0.1 mM EDTA and 0.1 M perchloric acid were added to the supernatant to inhibit DA degradation. Non-encapsulated cell aggregates cultured on a LA/PLO-coated dish were used as a positive control. A TSK-GEL Super-ODS column (100 × 4.6 mm; TOSOH, Tokyo, Japan) and an EC8020 electrochemical detector (TOSOH) were used to determine DA concentrations in the supernatant. The mobile phase was composed of 0.1 M citrate buffer solution (pH 2.5), 0.1 mM EDTA, 5 mM 1-octasulfonate, and 3% (v/v) methanol. The flow rate of the mobile phase was 1.2 mL/min. 2.6. Immunofluorescence examination Antibodies against octamer-binding transcription factor 3/4 (Oct3/4, 1:50, rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Nanog (1:200, rabbit monoclonal, Cell Signaling Technologies, Inc., MA, USA), stage-specific embryonic antigen-4 (SSEA-4, 1:200, mouse monoclonal, Millipore), β-tubulin III (1:500, rabbit monoclonal, Covance, Princeton, NJ, USA), and TH (1:200, mouse monoclonal, Millipore, and 1:200, mouse monoclonal, Covance) were used for immunohistochemistry. Cell aggregates were fixed with 4% paraformaldehyde in PBS for 1 h at 4 °C and then sequentially soaked in 5%, 10%, and 20% sucrose in PBS for 12 h at 4 °C. The cell aggregates then were embedded in Tissue-Tek (Sakura Finetechnical Co. Ltd., Tokyo, Japan) and frozen. Frozen specimens of 10 μm thickness were prepared. The specimens were treated with 0.2% Triton™ X-100 solution for 15 min at room temperature to permeabilize the cells and then with Blocking One (Nacalai Tesque) for 90 min to block nonspecific adsorption of antibodies. Antibody solutions were applied to the specimens and incubated for 1 h at room temperature. After being washed with PBS containing 0.05% Tween 20, the specimens were treated with Alexa Fluor 594 antimouse IgG and Alexa Fluor 488 anti-rabbit IgG (Invitrogen) at a dilution of 1:500 for 1 h at room temperature, then washed with PBS containing 0.05% Tween 20. The cell nuclei were counterstained with 1 μg/mL Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan), and the localization of secondary antibodies was analyzed under a fluorescent microscope (BX51 TRF, Olympus Optical Co., Ltd., Tokyo, Japan). For determining the percentage of TH-positive cells, cells were carefully examined on merged images enlarged with computer software. Cell
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numbers were counted at four sites on the same sample, and these data were averaged.
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3. Results 3.1. Microencapsulation and release of cell aggregates
2.7. Transplantation All animal experiments were carried out according to the guidelines of the Animal Welfare Committee of our Institute. Adult (8 weeks old) male Sprague–Dawley rats were housed under a 12-h light/12-h dark cycle with ad libitum access to food and water. Cell transplantation experiments were carried out with animals under anesthesia with pentobarbital sodium salt (50 mg/kg weight). A suspension of cell aggregates (50 cell aggregates, approximately 2.0 × 103 cells/aggregate, 5 μL) in PBS was stereotactically infused into the right or left striatum (coordinates 0.5 mm anterior and 2.0 mm lateral to the bregma, and 5.0 mm ventral) using a 10-μL Hamilton microsyringe. The microsyringe was first plunged 5.0 mm below the dural surface and immediately withdrawn 1.5 mm just before injection to avoid back-flow. Injections were performed at a rate of 1 μL/min. A total of 100 mg/kg of cyclosporine A (Novartis International AG, Basel, Switzerland) was intraperitoneally injected into the rats every day for immunosuppression. Rats were sacrificed at one week after transplantation and transcardially infused with PBS, followed by 4% paraformaldehyde for fixation. Brains were retrieved from animals and soaked in 4% paraformaldehyde for another 24 h at 4 °C. The brain tissue was embedded in paraffin and cut into 10 μm-thick horizontal sections using a microtome. After deparaffinization and antigen retrieval, the sections were immunologically stained as described in Section 2.6.
HiPS cells were differentiated into DA neuron precursors under the cell adherent culture as previously reported [4]. In this study, hiPS cells were also differentiated into the DA neural lineage (Fig. S1). The cells on day 18 were collected, and 6000 cells were transferred to each well of a U-bottom 96-well plate to allow formation of cell aggregates. Cell aggregates of about 300–400 μm in diameter were formed during an additional 5 days of culture (Fig. 1A). Supplemental Fig. 2 shows the immunofluorescent microphotographs of aggregates on day 23, it was possible to observe that the obtained aggregates also did not present cells positive for pluripotent markers such as Oct 3/4 and SSEA-4, and that majority of cells were β-tubulin III positive. The aggregates were encapsulated into alginate microbeads on day 23 (Fig. 1B). The size of the alginate–Ca2+ capsule was approximately 2 mm in diameter. Two or three cell aggregates were encapsulated in a capsule. The encapsulated aggregates were maintained for an additional 25 days to allow cell maturation (Fig. 1C), and the aggregates were successfully released from the alginate beads by treatment with 50 mM citrate buffer solution (Fig. 1D). No clear morphological difference, such as size of aggregates or their brightness, was observed between the aggregates on day 23 and those on day 48 (Fig. 1B and C). Hematoxylin and eosin staining of a thin section of an aggregate released from the alginate bead on day 48 is shown in Fig. 2A. No necrotic core was observed, indicating that the alginate bead allowed proper diffusion of nutrients and oxygen to the cells.
2.8. Statistical analysis
3.2. Immunohistological characterization of aggregates
Data are shown as mean ± standard deviation for at least three independent experiments. The data were compared using Student's t-tests. All statistical calculations were performed using JMP (SAS Institute Inc., NC, USA).
Immunofluorescence examination of aggregates that were released from beads on day 48 was carried out for a neuron marker, β-tubulin III, and a marker for DA neurons, TH. As shown in Fig. 2B, almost all cells in the released aggregates were β-tubulin positive. The percentage
Fig. 1. Alginate encapsulation and decapsulation. A: Phase-contrast micrographs of an aggregate of DA precursor cells on day 23. B: Phase-contrast micrograph of cell aggregates after encapsulation. Diameter capsule was approximately 2 mm. C: Phase-contrast micrograph of cell aggregates in microbeads on day 48. D: Phase-contrast micrograph of cell aggregates after removal of alginate–Ca2+ gel. Scale bars: 500 μm.
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Fig. 2. Immunofluorescent micrographs of cell aggregates on day 48. A: Hematoxylin and eosin staining of a thin section of a cell aggregate collected from a microbead on day 48. B–E: Fluorescent micrograph of cell aggregates immunologically stained using antibodies against β-tubulin III (green) and TH (red). Cell nuclei were stained with Hoechst 33258. F, G: Fluorescent micrograph of cell aggregates immunologically stained using antibodies against Oct3/4 (red) and Nanog (green). Cell nuclei were stained with Hoechst 33258. Scale bar: 100 μm.
of TH positive cells within the aggregates was 63 ± 13%. More than half of β-tubulin III-positive cells were TH positive, i.e., they were double positive for β-tubulin III and TH (Fig. 2B–E). Pluripotent stem cell markers such as Oct 3/4 and Nanog were not detected in immunofluorescent images of cell aggregates on day 48 (Fig. 2F and G). Aggregates released from the beads on day 48 were further characterized after being cultured on the top of a laminin/poly-L-ornithinecoated dish for an additional 7 days. The aggregates successfully adhered to the dish. Cells migrated out from the peripheral area of the aggregates and spread onto the dish surface (Fig. 3A). The cell extended long neurites and thus had an elongated morphology (Fig. 3B). The majority of cells that spread out from the aggregates were β-tubulin III positive (Fig. 3C), and a considerable percentage of cells were double positive for β-tubulin III and TH (Fig. 3D and E). 3.3. DA production Six hundred aggregates collected from microbeads were depolarized by exposure to 56 mM KCl to induce DA release. DA released from the cells into the medium was analyzed by HPLC, and Fig. 4A shows the representative chromatograms of this analysis. As a control, 600 aggregates were induced to adhere on the top of a laminin/poly-L-ornithine-coated dish on day 23 and cultured for additional days. The released amounts of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were also analyzed
under the same conditions. Although no DA was observed on day 25 from aggregates either enclosed in beads or maintained on a laminin/ poly-L-ornithine substrate, substantial amounts of DA and DOPAC were secreted from aggregates enclosed in the beads or maintained on the laminin/poly-L-ornithine substrate on day 48. These results suggest that the cell aggregates matured into DA-releasing cells during culture from day 25 to day 48. Amounts of DA release tended to gradually increase with culturing periods in both cases (Fig. S3). No significant difference in DA production was observed between aggregates in microbeads and aggregates cultured on the laminin/poly-L-ornithine substrate, as shown in Fig. 4B. 3.4. In vivo behavior of DA neurons matured in microbeads Aggregates in beads were cultured until day 41 and collected and transplanted into rat right or left striatum. Grafts were retrieved at one week post-transplantation. Immunofluorescence studies of thin sections of the grafts were carried out for human nuclei (hNu), βtubulin III, and TH (Fig. 5). Many hNu positive cells were clearly observed, and some were also positive for β-tubulin III and TH. In addition, the cells extended neurites from the graft into the host brain, indicating integration of neurons into host brain tissue. These results suggest that DA neurons in the aggregate can survive and actively migrate out from the aggregates into the host brain tissue.
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Fig. 3. Effect of decapsulation on DA neurons derived from hiPS cells. A, B: Phase-contrast micrographs of cells cultured on LA/PLO substrate for 7 days after retrieval from alginate beads. C–E: Immunofluorescent micrograph of cells cultured on LA/PLO substrate for 7 days after retrieval from alginate beads. Cell aggregates immunologically stained using antibodies against β-tubulin III (green) and TH (red). Scale bar: 100 μm.
4. Discussion PD is characterized by the selective and progressive loss of DA neurons. At early stages, the symptoms are motor related, i.e., patients usually present with muscle rigidity, tremor, and postural instability.
Fig. 4. DA production by cell aggregates retrieved from alginate beads. Six hundred cell aggregates were depolarized in 56 mM KCl for 30 min. The supernatants were analyzed using reverse-phase HPLC. As a positive control, the same number of cell aggregates was seeded onto LM/PLO substrate and cultured for 25 days to induce maturation of DA neurons. A: Representative chromatograms of a standard solution of 10 nM DA and 10 nM DOPAC (upper line), supernatant from decapsulated cell aggregates (middle line), and supernatant from cells cultured on LA/PLO substrate (bottom line). B: The means of DA amount released from encapsulated cell aggregates (n = 5) and cells cultured on LA/PLO substrate (n = 4).
Non-motor-related symptoms such as cognitive impairment arise as the pathology progresses [18, 19]. Although therapies, including administration of DA agonists and deep brain stimulation, have been applied to treat PD patients and show some effectiveness, this effectiveness decreases with time [20, 21]. Cell replacement therapy has been proposed as an alternative method for treating PD patients. Pluripotent cells such as ES and iPS cells have been identified as a suitable source of DA neurons for this cell replacement therapy, and several protocols for preparing DA neurons from ES/iPS cells have been reported [2–4]. In most of the methods, pluripotent stem cells are differentiated into DA neurons under an adherent culture. Releasing the adhered cells from culture dishes requires enzymatic treatment, which may damage the cells and decrease the yield of DA neurons [22]. In addition, large numbers of DA neurons are needed for clinical application. In our previous study [7], we differentiated hiPS cells into DA precursors under a cell adherent culture, and aggregates of DA precursors were then encapsulated in agarose microbeads. The agarose effectively protected cells from mechanical stress, and the aggregates thus were maintained for a sufficiently long period to allow DA precursors to mature into DA neurons. However, the agarose shell removal was done under a microscope using a scalpel to collect DA neurons, which is impractical if a large amount of aggregates is needed. Therefore, in this study, we evaluated alginate for enclosing aggregates of DA precursors in microbeads. Ca2+-crosslinked alginate has been widely used to microencapsulate various types of cells [12, 13, 23, 24] and to encapsulate growth factors or cytokines as a drug delivery system [25, 26]. A minor difference in the stem or precursor cell culture medium can drastically affect cell fate. In these alginate–Ca2+ microbeads, cells are exposed to dense anionic functional groups, carboxyl side groups of alginate, and low Ca2+ concentration, an ionic environment that might affect their fate, survival, and differentiation. We found, however, that DA precursors maintained their viability and effectively differentiated into TH-positive neurons. Aggregates that were enclosed in alginate–Ca2+ microbeads and maintained over 25 days could release DA at the same level as those maintained on culture dishes without encapsulation (positive control). On day 23, majority of cells were positive for β-tubulin III (Fig. S2), at this stage less than 10% of cells, however, were positive for
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Fig. 5. Transplantation of DA neurons prepared in an alginate capsule. Cell aggregates on day 41 were transplanted into immunosuppressed Sprague–Dawley rats. At 7 days from injection, animals were sacrificed. A–F: Collected tissue was immunologically stained with antibodies against β-tubulin III (A) and TH (D). Human nuclei-positive cells showed transplanted cells (B) and (E). (C) and (F): merge. Scale bar: 100 μm.
TH. In our previous study, it was demonstrated that dopamine release continuously increased during 48 days of culture [7]. In this study, more than 60% of cells were positive for TH on day 48 (Fig. 2). No clear difference in aggregates size was observed when comparing day 23 aggregates with day 48 aggregates. This might be due to the absence of cells which can proliferate, such as pluripotent stem cells on day 23 and day 48. In the case of aggregates cultured on the top of a PLO/laminin coated dish, cells migrated from the aggregate and propagated radially through the dish surface, but not proliferated. In addition, DA neuron aggregates could be easily released from microbeads by exposure of the microbeads to a citrate solution. Alginate–Ca2+ microbeads thus can evade the release difficulty we encountered previously in the case of agarose microbeads, and the alginate microbead is suitable for maintaining DA precursor aggregates for a long period. Our preliminary in vivo study showed viable cells in the rat brain graft with cells extending neurites into the host tissue at 7 days posttransplantation. This result indicates graft integration with the host tissue. This part of the study was focused on determining how aggregates would respond when transplanted into the brain and to verify graft survival after one week. More detailed study is planned of graft outcomes in a rat PD model, addressing long-term graft survival, motor recovery, and tumor formation. 5. Conclusion Cell aggregates encapsulated in alginate–Ca2 + beads successfully differentiated into mature DA neurons. Under treatment with a chelating agent, the cell aggregates were easily retrieved from the beads without damage. In a transplantation study, DA neurons prepared in alginate–Ca2 + beads integrated with the host brain. The alginate– Ca2+ bead can provide a suitable environment for preparing DA neurons on a large scale. Transparency document The Transparency document associated with this article can be found, in the online version.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen
Maturation of human iPS cell-derived dopamine neuron precursors in alginate–Ca2 + hydrogel Mitsue Komatsu, Shuhei Konagaya, Edgar Y. Egawa, Hiroo Iwata ⁎ Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 14 April 2015 Accepted 27 April 2015 Available online 4 May 2015 Keywords: iPS cell Dopamine Neuron precursor Alginate Microencapsulation
a b s t r a c t Background: Pluripotent stem cells (embryonic stem/induced pluripotent stem cells) have been widely studied as a potential cell source for cell transplantation therapy of Parkinson's disease. However, some difficulties remain to be overcome. These include the need to prepare a large number of dopamine (DA) neurons for clinical use and to culture the cells for a long period to allow their functional maturation and the removal of undifferentiated cells. Methods: In this study, aggregates of DA neuron precursors were enclosed in alginate–Ca2+ microbeads, and the encapsulated aggregates were cultured for 25 days to induce cell maturation. Results: More than 60% of cells in the aggregates differentiated into tyrosine hydroxylase-positive DA neurons. The aggregates could release DA at the same level as aggregates maintained on culture dishes without encapsulation. In addition, by exposure to a citrate solution, the alginate–Ca2+ gel layer could be easily removed from aggregates without damaging the DA neurons. When the aggregates were transplanted into rat brain, viable cells were found in the graft at one week post-transplantation, with cells extending neurites into the host tissue. Conclusions: Cell aggregates encapsulated in alginate–Ca2+ beads successfully differentiated into mature DA neurons. General significance: The alginate–Ca2+ microbead is suitable for maintaining DA precursor aggregates for a long period to allow their functional maturation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pluripotent stem cells, such as embryonic stem (ES) and induced pluripotent stem (iPS) cells, can be expanded without limit and induced to differentiate into multiple functional cell types and have been considered as potential cell sources for cell transplantation therapy of Parkinson's disease (PD) [1]. Protocols for differentiation of pluripotent cells into dopamine (DA) neurons have been widely studied [2–4], and differentiated DA neurons from ES cells or iPS cells have been transplanted into PD model animals to demonstrate efficacy [5, 6]. Although DA neurons derived from human pluripotent stem cells are being considered as treatment for PD patients in several countries, some difficulties remain to be overcome. One of these is that huge numbers of DA neurons must be prepared for clinical application because the human body is about 3000 times larger than that of a mouse. Another problem requiring careful consideration with transplantation of iPSderived cells is tumor formation [8, 9] because undifferentiated cells contaminating grafts might form tumors. It has been claimed that the risk of tumor formation can be reduced by maturing cells for a long period of culture in vitro before transplantation [10, 11].
⁎ Corresponding author. Tel.: +81 75 751 4119; fax: +81 75 751 4646. E-mail address: [email protected] (H. Iwata).
http://dx.doi.org/10.1016/j.bbagen.2015.04.011 0304-4165/© 2015 Elsevier B.V. All rights reserved.
In our previous studies [7], we examined microencapsulation of DA neuron precursors in agarose beads as a way to overcome these two difficulties. In that work [7], DA neuron precursors effectively matured by long-term culture in agarose beads, and the cells released DA for more than 40 days. In addition, microbeads containing cells could be cryopreserved. Although agarose microencapsulation provides a good supporting environment for the preparation and storage of DA neurons, the tedious process of removal of agarose microbeads by hand is required before transplantation to allow their direct interaction with host tissue. In the current study, we evaluated alginate–Ca2+ hydrogel for microencapsulation of DA neuron precursors. An alginate solution turns into gel when it is exposed to a solution of a divalent metal ion such as Ca2 + and Ba2 +. Alginate–Ca2 + hydrogels have been widely examined for preparation of a bioartificial pancreas, specifically microencapsulated islets of Langerhans [12, 13]. The gel can be returned to solution by the addition of chelating agents, such as citric acid or ethylenediamine-tetraacetic acid (EDTA). It has been reported that cells are easily encapsulated in alginate–Ca2+ gel and can be retrieved undamaged from the capsule under moderate conditions [14]. Alginate–Ca2+ thus seems to be a suitable material for microencapsulation of DA neuron precursors and their long-term culture. Sometimes, a minor difference in cell culture medium, however, drastically affects cell fate, such as survival and direction of differentiation. In this study,
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aggregates of DA neuron precursors were enclosed in alginate–Ca2 + microbeads and cultured for a long period. In an alginate–Ca2 + microbead, cells are exposed to dense anionic functional groups, carboxyl side groups of alginate, and low Ca2+ concentration. Their effects on the fate of DA neuron precursors, number of tyrosine hydroxylase (TH)-positive cells, and DA release function were examined.
suspension was dropped through a 25-G hypodermic needle into 100 mM CaCl2 solution containing 2 mM KCl and 1 mM HEPES. After 10 min for complete gelation, the alginate microbeads were washed twice with physiological saline. The encapsulated cell aggregates were cultured in the basal medium for an additional 25 days for maturation into DA neurons. Culture medium was replaced with fresh medium every 2 or 3 days.
2. Materials and methods 2.1. Human iPS (hiPS) cell culture and differentiation into DA neurons Two lines of hiPS cells (201B7 [15] and 253G1 [16], RIKEN Cell Bank, Ibaraki, Japan) were used in this study. Undifferentiated hiPS cells were maintained on an SNL 76/7 (ECACC, Salisbury, UK) feeder layer as previously described [17]. HiPS cells were differentiated into the DA neuronal lineage following the protocol shown in Scheme 1 [7]. Briefly, undifferentiated hiPS cells were seeded on Matrigel (BD Bioscience, San Jose, CA, USA) and cultured in medium containing several cytokines and small molecules. Differentiation medium was changed daily, and on day 18, subpopulations of cells were used for microencapsulation. The remaining cells were subcultured on a laminin/poly-L-ornithine (LA/ PLO)-coated dish for additional days. 2.2. Formation of cell aggregates On day 18, cells were detached from culture dishes by treatment with Accumax (Innovative Cell Technologies, Inc., San Diego, CA, USA) for 3 min. The cells were suspended in DMEM/F12 supplemented with 2.5 mM GlutaMAX, 2% B27 supplement (Invitrogen), 10 ng/mL brainderived neurotrophic growth factor (Wako Pure Chemical Industries, Osaka, Japan), 10 ng/mL glial cell line-derived neurotrophic growth factor (Wako), 0.2 mM ascorbic acid (Nacalai Tesque), 0.5 mM dibutyryl cyclic AMP (dcAMP, Nacalai Tesque), 1 ng/mL transforming growth factor-β3 (TGF-β3, R&D Systems), 100 U/mL penicillin, and 100 μg/mL streptomycin. This medium is referred to hereafter as the basal medium. U-bottom 96-well plates were pre-coated with 2% Pluronic® F127 overnight to inhibit cell adhesion. After plating, the cells were washed five times with phosphate buffered saline (PBS), applied to wells at a density of 6000 cells/well, and centrifuged at 1000 rpm for 3 min. The cells were cultured in an incubator at 37 °C in 5% CO2 atmosphere for 5 days to prepare cell aggregates. 2.3. Encapsulation Cell aggregates were enclosed in alginate–Ca2 + beads. Approximately 1000 cell aggregates were mixed with 2 mL of 1% alginate (Mw; 3 × 105, Nacalai Tesque) in Krebs–Ringer/HEPES solution. The
Scheme 1. Experimental protocol. HiPS cells were differentiated into DA neurons in a series of media supplemented with different inhibitors and cytokines. DA precursor cells were collected from the dish at day 18, and 6000 cells were applied to each well of 96well U-bottom plates that were pretreated with Pluronic® 127F. The cells were cultured for an additional 5 days to form cell aggregates, which were then encapsulated in alginate beads on day 23. Encapsulated cell aggregates were cultured in the same medium type as that used for aggregate formation. On day 48, alginate hydrogel was dissolved by exposure to a citrate buffer solution, and cell aggregates were collected.
2.4. Decapsulation On day 48, alginate–Ca2+ beads were dissolved to collect cell aggregates. After being washed with saline solution three times, the alginate– Ca2+ beads were treated with 50 mM citrate buffer solution in physiological saline solution for 15 min at 37 °C. The cell aggregates were washed two times with PBS and then seeded onto a LM/PLO-coated dish and cultured for one week. The cell aggregates that adhered on the LM/PLO-coated dish were subjected to immunofluorescence examination. 2.5. DA secretion Six hundred decapsulated cell aggregates were washed twice with PBS supplemented with 0.33 mM Mg2+ and 0.9 mM Ca2+. The cells were incubated for 30 min in 56 mM KCl/Hanks balanced salt solution supplemented with 0.33 mM Mg2+ and 0.9 mM Ca2+ to induce depolarization of the cell membrane. A total of 300 μL of the supernatant was collected into a test tube, and 0.1 mM EDTA and 0.1 M perchloric acid were added to the supernatant to inhibit DA degradation. Non-encapsulated cell aggregates cultured on a LA/PLO-coated dish were used as a positive control. A TSK-GEL Super-ODS column (100 × 4.6 mm; TOSOH, Tokyo, Japan) and an EC8020 electrochemical detector (TOSOH) were used to determine DA concentrations in the supernatant. The mobile phase was composed of 0.1 M citrate buffer solution (pH 2.5), 0.1 mM EDTA, 5 mM 1-octasulfonate, and 3% (v/v) methanol. The flow rate of the mobile phase was 1.2 mL/min. 2.6. Immunofluorescence examination Antibodies against octamer-binding transcription factor 3/4 (Oct3/4, 1:50, rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Nanog (1:200, rabbit monoclonal, Cell Signaling Technologies, Inc., MA, USA), stage-specific embryonic antigen-4 (SSEA-4, 1:200, mouse monoclonal, Millipore), β-tubulin III (1:500, rabbit monoclonal, Covance, Princeton, NJ, USA), and TH (1:200, mouse monoclonal, Millipore, and 1:200, mouse monoclonal, Covance) were used for immunohistochemistry. Cell aggregates were fixed with 4% paraformaldehyde in PBS for 1 h at 4 °C and then sequentially soaked in 5%, 10%, and 20% sucrose in PBS for 12 h at 4 °C. The cell aggregates then were embedded in Tissue-Tek (Sakura Finetechnical Co. Ltd., Tokyo, Japan) and frozen. Frozen specimens of 10 μm thickness were prepared. The specimens were treated with 0.2% Triton™ X-100 solution for 15 min at room temperature to permeabilize the cells and then with Blocking One (Nacalai Tesque) for 90 min to block nonspecific adsorption of antibodies. Antibody solutions were applied to the specimens and incubated for 1 h at room temperature. After being washed with PBS containing 0.05% Tween 20, the specimens were treated with Alexa Fluor 594 antimouse IgG and Alexa Fluor 488 anti-rabbit IgG (Invitrogen) at a dilution of 1:500 for 1 h at room temperature, then washed with PBS containing 0.05% Tween 20. The cell nuclei were counterstained with 1 μg/mL Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan), and the localization of secondary antibodies was analyzed under a fluorescent microscope (BX51 TRF, Olympus Optical Co., Ltd., Tokyo, Japan). For determining the percentage of TH-positive cells, cells were carefully examined on merged images enlarged with computer software. Cell
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numbers were counted at four sites on the same sample, and these data were averaged.
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3. Results 3.1. Microencapsulation and release of cell aggregates
2.7. Transplantation All animal experiments were carried out according to the guidelines of the Animal Welfare Committee of our Institute. Adult (8 weeks old) male Sprague–Dawley rats were housed under a 12-h light/12-h dark cycle with ad libitum access to food and water. Cell transplantation experiments were carried out with animals under anesthesia with pentobarbital sodium salt (50 mg/kg weight). A suspension of cell aggregates (50 cell aggregates, approximately 2.0 × 103 cells/aggregate, 5 μL) in PBS was stereotactically infused into the right or left striatum (coordinates 0.5 mm anterior and 2.0 mm lateral to the bregma, and 5.0 mm ventral) using a 10-μL Hamilton microsyringe. The microsyringe was first plunged 5.0 mm below the dural surface and immediately withdrawn 1.5 mm just before injection to avoid back-flow. Injections were performed at a rate of 1 μL/min. A total of 100 mg/kg of cyclosporine A (Novartis International AG, Basel, Switzerland) was intraperitoneally injected into the rats every day for immunosuppression. Rats were sacrificed at one week after transplantation and transcardially infused with PBS, followed by 4% paraformaldehyde for fixation. Brains were retrieved from animals and soaked in 4% paraformaldehyde for another 24 h at 4 °C. The brain tissue was embedded in paraffin and cut into 10 μm-thick horizontal sections using a microtome. After deparaffinization and antigen retrieval, the sections were immunologically stained as described in Section 2.6.
HiPS cells were differentiated into DA neuron precursors under the cell adherent culture as previously reported [4]. In this study, hiPS cells were also differentiated into the DA neural lineage (Fig. S1). The cells on day 18 were collected, and 6000 cells were transferred to each well of a U-bottom 96-well plate to allow formation of cell aggregates. Cell aggregates of about 300–400 μm in diameter were formed during an additional 5 days of culture (Fig. 1A). Supplemental Fig. 2 shows the immunofluorescent microphotographs of aggregates on day 23, it was possible to observe that the obtained aggregates also did not present cells positive for pluripotent markers such as Oct 3/4 and SSEA-4, and that majority of cells were β-tubulin III positive. The aggregates were encapsulated into alginate microbeads on day 23 (Fig. 1B). The size of the alginate–Ca2+ capsule was approximately 2 mm in diameter. Two or three cell aggregates were encapsulated in a capsule. The encapsulated aggregates were maintained for an additional 25 days to allow cell maturation (Fig. 1C), and the aggregates were successfully released from the alginate beads by treatment with 50 mM citrate buffer solution (Fig. 1D). No clear morphological difference, such as size of aggregates or their brightness, was observed between the aggregates on day 23 and those on day 48 (Fig. 1B and C). Hematoxylin and eosin staining of a thin section of an aggregate released from the alginate bead on day 48 is shown in Fig. 2A. No necrotic core was observed, indicating that the alginate bead allowed proper diffusion of nutrients and oxygen to the cells.
2.8. Statistical analysis
3.2. Immunohistological characterization of aggregates
Data are shown as mean ± standard deviation for at least three independent experiments. The data were compared using Student's t-tests. All statistical calculations were performed using JMP (SAS Institute Inc., NC, USA).
Immunofluorescence examination of aggregates that were released from beads on day 48 was carried out for a neuron marker, β-tubulin III, and a marker for DA neurons, TH. As shown in Fig. 2B, almost all cells in the released aggregates were β-tubulin positive. The percentage
Fig. 1. Alginate encapsulation and decapsulation. A: Phase-contrast micrographs of an aggregate of DA precursor cells on day 23. B: Phase-contrast micrograph of cell aggregates after encapsulation. Diameter capsule was approximately 2 mm. C: Phase-contrast micrograph of cell aggregates in microbeads on day 48. D: Phase-contrast micrograph of cell aggregates after removal of alginate–Ca2+ gel. Scale bars: 500 μm.
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Fig. 2. Immunofluorescent micrographs of cell aggregates on day 48. A: Hematoxylin and eosin staining of a thin section of a cell aggregate collected from a microbead on day 48. B–E: Fluorescent micrograph of cell aggregates immunologically stained using antibodies against β-tubulin III (green) and TH (red). Cell nuclei were stained with Hoechst 33258. F, G: Fluorescent micrograph of cell aggregates immunologically stained using antibodies against Oct3/4 (red) and Nanog (green). Cell nuclei were stained with Hoechst 33258. Scale bar: 100 μm.
of TH positive cells within the aggregates was 63 ± 13%. More than half of β-tubulin III-positive cells were TH positive, i.e., they were double positive for β-tubulin III and TH (Fig. 2B–E). Pluripotent stem cell markers such as Oct 3/4 and Nanog were not detected in immunofluorescent images of cell aggregates on day 48 (Fig. 2F and G). Aggregates released from the beads on day 48 were further characterized after being cultured on the top of a laminin/poly-L-ornithinecoated dish for an additional 7 days. The aggregates successfully adhered to the dish. Cells migrated out from the peripheral area of the aggregates and spread onto the dish surface (Fig. 3A). The cell extended long neurites and thus had an elongated morphology (Fig. 3B). The majority of cells that spread out from the aggregates were β-tubulin III positive (Fig. 3C), and a considerable percentage of cells were double positive for β-tubulin III and TH (Fig. 3D and E). 3.3. DA production Six hundred aggregates collected from microbeads were depolarized by exposure to 56 mM KCl to induce DA release. DA released from the cells into the medium was analyzed by HPLC, and Fig. 4A shows the representative chromatograms of this analysis. As a control, 600 aggregates were induced to adhere on the top of a laminin/poly-L-ornithine-coated dish on day 23 and cultured for additional days. The released amounts of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were also analyzed
under the same conditions. Although no DA was observed on day 25 from aggregates either enclosed in beads or maintained on a laminin/ poly-L-ornithine substrate, substantial amounts of DA and DOPAC were secreted from aggregates enclosed in the beads or maintained on the laminin/poly-L-ornithine substrate on day 48. These results suggest that the cell aggregates matured into DA-releasing cells during culture from day 25 to day 48. Amounts of DA release tended to gradually increase with culturing periods in both cases (Fig. S3). No significant difference in DA production was observed between aggregates in microbeads and aggregates cultured on the laminin/poly-L-ornithine substrate, as shown in Fig. 4B. 3.4. In vivo behavior of DA neurons matured in microbeads Aggregates in beads were cultured until day 41 and collected and transplanted into rat right or left striatum. Grafts were retrieved at one week post-transplantation. Immunofluorescence studies of thin sections of the grafts were carried out for human nuclei (hNu), βtubulin III, and TH (Fig. 5). Many hNu positive cells were clearly observed, and some were also positive for β-tubulin III and TH. In addition, the cells extended neurites from the graft into the host brain, indicating integration of neurons into host brain tissue. These results suggest that DA neurons in the aggregate can survive and actively migrate out from the aggregates into the host brain tissue.
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Fig. 3. Effect of decapsulation on DA neurons derived from hiPS cells. A, B: Phase-contrast micrographs of cells cultured on LA/PLO substrate for 7 days after retrieval from alginate beads. C–E: Immunofluorescent micrograph of cells cultured on LA/PLO substrate for 7 days after retrieval from alginate beads. Cell aggregates immunologically stained using antibodies against β-tubulin III (green) and TH (red). Scale bar: 100 μm.
4. Discussion PD is characterized by the selective and progressive loss of DA neurons. At early stages, the symptoms are motor related, i.e., patients usually present with muscle rigidity, tremor, and postural instability.
Fig. 4. DA production by cell aggregates retrieved from alginate beads. Six hundred cell aggregates were depolarized in 56 mM KCl for 30 min. The supernatants were analyzed using reverse-phase HPLC. As a positive control, the same number of cell aggregates was seeded onto LM/PLO substrate and cultured for 25 days to induce maturation of DA neurons. A: Representative chromatograms of a standard solution of 10 nM DA and 10 nM DOPAC (upper line), supernatant from decapsulated cell aggregates (middle line), and supernatant from cells cultured on LA/PLO substrate (bottom line). B: The means of DA amount released from encapsulated cell aggregates (n = 5) and cells cultured on LA/PLO substrate (n = 4).
Non-motor-related symptoms such as cognitive impairment arise as the pathology progresses [18, 19]. Although therapies, including administration of DA agonists and deep brain stimulation, have been applied to treat PD patients and show some effectiveness, this effectiveness decreases with time [20, 21]. Cell replacement therapy has been proposed as an alternative method for treating PD patients. Pluripotent cells such as ES and iPS cells have been identified as a suitable source of DA neurons for this cell replacement therapy, and several protocols for preparing DA neurons from ES/iPS cells have been reported [2–4]. In most of the methods, pluripotent stem cells are differentiated into DA neurons under an adherent culture. Releasing the adhered cells from culture dishes requires enzymatic treatment, which may damage the cells and decrease the yield of DA neurons [22]. In addition, large numbers of DA neurons are needed for clinical application. In our previous study [7], we differentiated hiPS cells into DA precursors under a cell adherent culture, and aggregates of DA precursors were then encapsulated in agarose microbeads. The agarose effectively protected cells from mechanical stress, and the aggregates thus were maintained for a sufficiently long period to allow DA precursors to mature into DA neurons. However, the agarose shell removal was done under a microscope using a scalpel to collect DA neurons, which is impractical if a large amount of aggregates is needed. Therefore, in this study, we evaluated alginate for enclosing aggregates of DA precursors in microbeads. Ca2+-crosslinked alginate has been widely used to microencapsulate various types of cells [12, 13, 23, 24] and to encapsulate growth factors or cytokines as a drug delivery system [25, 26]. A minor difference in the stem or precursor cell culture medium can drastically affect cell fate. In these alginate–Ca2+ microbeads, cells are exposed to dense anionic functional groups, carboxyl side groups of alginate, and low Ca2+ concentration, an ionic environment that might affect their fate, survival, and differentiation. We found, however, that DA precursors maintained their viability and effectively differentiated into TH-positive neurons. Aggregates that were enclosed in alginate–Ca2+ microbeads and maintained over 25 days could release DA at the same level as those maintained on culture dishes without encapsulation (positive control). On day 23, majority of cells were positive for β-tubulin III (Fig. S2), at this stage less than 10% of cells, however, were positive for
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Fig. 5. Transplantation of DA neurons prepared in an alginate capsule. Cell aggregates on day 41 were transplanted into immunosuppressed Sprague–Dawley rats. At 7 days from injection, animals were sacrificed. A–F: Collected tissue was immunologically stained with antibodies against β-tubulin III (A) and TH (D). Human nuclei-positive cells showed transplanted cells (B) and (E). (C) and (F): merge. Scale bar: 100 μm.
TH. In our previous study, it was demonstrated that dopamine release continuously increased during 48 days of culture [7]. In this study, more than 60% of cells were positive for TH on day 48 (Fig. 2). No clear difference in aggregates size was observed when comparing day 23 aggregates with day 48 aggregates. This might be due to the absence of cells which can proliferate, such as pluripotent stem cells on day 23 and day 48. In the case of aggregates cultured on the top of a PLO/laminin coated dish, cells migrated from the aggregate and propagated radially through the dish surface, but not proliferated. In addition, DA neuron aggregates could be easily released from microbeads by exposure of the microbeads to a citrate solution. Alginate–Ca2+ microbeads thus can evade the release difficulty we encountered previously in the case of agarose microbeads, and the alginate microbead is suitable for maintaining DA precursor aggregates for a long period. Our preliminary in vivo study showed viable cells in the rat brain graft with cells extending neurites into the host tissue at 7 days posttransplantation. This result indicates graft integration with the host tissue. This part of the study was focused on determining how aggregates would respond when transplanted into the brain and to verify graft survival after one week. More detailed study is planned of graft outcomes in a rat PD model, addressing long-term graft survival, motor recovery, and tumor formation. 5. Conclusion Cell aggregates encapsulated in alginate–Ca2 + beads successfully differentiated into mature DA neurons. Under treatment with a chelating agent, the cell aggregates were easily retrieved from the beads without damage. In a transplantation study, DA neurons prepared in alginate–Ca2 + beads integrated with the host brain. The alginate– Ca2+ bead can provide a suitable environment for preparing DA neurons on a large scale. Transparency document The Transparency document associated with this article can be found, in the online version.
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