Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Abstract

Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson's disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues.

Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application.

Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo.

Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior.

Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

Keywords: Cryopreservation; Parkinson’s disease; cell-based therapy; dopaminergic neuron; induced pluripotent stem cells; neurosphere.

Fig. 1

Schematic overview of the protocol steps. Cryopreserved×1 and Cryopreserved×2 are defined in the main text. iPSCs, induced pluripotent stem cells.

Fig. 2

Effects of cryopreservation media on iPSC-derived neurospheres (A): Viability and (B): neurite extension of spheres from unsorted cells cryopreserved at –0.5°C/min on day 28 using the cryopreservation media shown in table 1 (n = 4). Viability and neurite extensions were analyzed on day 1 (24 hours) and day 5, respectively. One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001 versus Bambanker hRM. C: Immunostaining of neurites for PSA-NCAM. Scale bars, 1 mm. Data are shown as means±SD.

Fig. 3

Time-temperature curves of the sample (red line), freezing chamber (blue line), and program (gray line). Bambanker hRM was used as the sample. The temperature change caused by latent heat release is magnified in the lower graphs.

Fig. 4

Effects of the freezing program and equilibration time on iPSC-derived neurospheres. A, C) Viability and (B, D) neurite extensions of spheres from unsorted cells cryopreserved on day 28 under the different freezing programs after 15 min (A, B) and 60 min (C, D) equilibration in Bambanker hRM (n = 4). Viability and neurite extensions were analyzed on day 1 (24 h) and day 5, respectively. One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 versus Proton Freezer. Data are shown as means±SD.

Fig. 5

Characterization of cryopreserved spheres derived from S17 in vitro. A) Immunostaining of the spheres on day 35. FOXA2/DAPI (left), NURR1/TH (center), and SOX1/KI67/PAX6/DAPI (right). Scale bars 100μm. B, C) The percentages of FOXA2⁺, NURR1⁺, TH⁺ (B) and SOX1⁺, PAX6⁺ and KI67⁺ (C) cells per total cells on day 35 (n = 4). D, E) Gene expressions of the spheres relative to GAPDH measured by quantitative RT-PCR (n = 4). D28, cells cultured for 28 days; D28 + 7, cells cultured for 7 days after 28 days cryofreezing; D35, fresh cells cultured for 35 days. The expression level of D28 (D) and undifferentiated cells (D0) (E) was set to 1. There were no significant differences between D35 and D28 + 7 by one-way ANOVA with Tukey’s multiple comparisons test (D). One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^****p < 0.0001 versus D0 (E). F) Immunostaining of post-thawed iPSC-derived DA neurons for TUBB3, TH, and FOXA2 on day 49. Scale bars, 50μm. G) Representative induced action potentials of post-thawed iPSC-derived DA neurons on day 68. H) Box plots display the value in each sphere of spike amplitudes on day 42. I) The dopamine release on day 56 induced by high potassium stimulation (n = 4). Data are shown as means±SD.

Fig. 6

Graft survival and function of cryopreserved spheres. A) The methamphetamine-induced rotations of rats that received the grafts. (n = 4–7). Two-way ANOVA with Tukey’s multiple comparisons test; ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 versus vehicle group. B) Immunostaining of representative grafts for HNA (green) and DAPI (blue). C) DAB staining of representative grafts for TH. The right panels are magnified images of the left panels. Scale bars, 50μm. D) The number of survived HNA⁺ cells in the grafts (n = 4–7). E) Number of survived TH⁺ cells in the grafts (n = 4–7). F) The percentages of TH⁺ cells per survived human cells (n = 4–7). One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^**p < 0.01 (D–F). G) Immunostaining of the grafts derived from cryopreserved cells for FOXA2, TH, and HNA (upper) and GIRK2, CALB, and TH (lower). Scale bars, 50μm. Data are shown as mean±SD.